ES2739082T3 - In vitro differentiation of pluripotent stem cells to pancreatic endodermal cells (PEC) and endocrine cells - Google Patents

Abstract

A method of producing an unipotent human immature beta cell comprising: contacting cells of the definitive human endodermal lineage in vitro with a member of the TGFβ superfamily and a member of the Wnt family, thus producing a unipotent human immature beta cell.

Description

DESCRIPTION

In vitro differentiation of pluripotent stem cells to pancreatic endodermal cells (PEC) and endocrine cells

Field of the Invention

The present invention relates, in general, to the isolation, maintenance and use of cell cultures. More specifically, it refers to cell compositions and methods for differentiating pluripotent stem cells in vitro to cultures of pancreatic endodermal cells (PEC) enriched in a non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-), but relatively replete with cells compromised with the endocrine lineage (CHGA +). The specification also describes, for the first time, cellular compositions and methods for differentiating pluripotent stem cells in vitro to endocrine cells that, after implantation in a mammal, produce insulin in response to glucose stimulation in vivo. The specification also describes cell compositions and methods for differentiating pluripotent stem cells in vitro to endocrine cells that produce insulin in vitro.

Background

The development of expanded populations of human pancreatic p cells that can be used for cell transplantation is an important goal of diabetes research. In recent years, attention has focused on the use of pluripotent stem cells as a possible renewable source for p cells. Due to the complex nature of pancreatic endocrine differentiation, which, at present, has not yet been elucidated, the differentiation of pluripotent stem cells to mature and functional p cells has not been effectively achieved in vitro. Until now, only progenitor cells, such as pancreatic endoderm cells (PEC), have been transplanted to rodents and, after 8 to 12 weeks, demonstrate the function of human-specific p cells. The PEC cell populations comprise at least two cell populations: a non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-) and cells committed to the endocrine lineage (CHGA +). It was discovered that, after PEC implantation, it is the non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-) that matures and forms the p cells in vivo. Transplantation of PEC cell populations requires a long maturation time in vivo (8-12 weeks) that can be shortened or eliminated if: (a) PEC populations in vitro contain more than the type of cells that mature in p cells in vivo (ie, non-endocrine multipotent pancreatic progenitor cells (CHGA-)), (b) terminal differentiation (p cell) is achieved in vitro; or (c) an in vitro progenitor population is identified that is farther along the path of differentiation than is currently used, that is, an advanced cell population.

Although the present application is not limited to any theory, a possible explanation for why pluripotent stem cells do not mature to become fully functional p cells in vitro is that in vitro derived cells do not have the same expression of markers at the same points. of time than during the in vivo pancreatic development of mammals. For example, the expression of NGN3 during differentiation in vitro occurs earlier than in pancreatic development of mammals in vivo. In fact, it has been observed that, in traditional 4-stage differentiation protocols, as described in the applicant's numerous publications and patents, PEC populations express NGN3 and NKX2.2. Therefore, the suppression or inhibition of the expression of NGN3 until after the expression of the differentiation of the multipotent non-endocrine pancreatic progenitor subpopulation PDX1 / NKX6.1 is an important step to achieve in vitro differentiation of pluripotent stem cells to mature and functional p cells or the differentiation of PEC populations that contain more non-endocrine multipotent pancreatic progenitor cells (CHGA-) than cells compromised with the endocrine lineage (CHGA +).

The identification of a protocol that delays the expression of NGN3 and NKX2.2 is not evident, because the differentiation protocol should not interrupt, and preferably increases, the formation of the non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-) and should minimize the cell loss; or at least maintain adequate cell mass and cell performance (i.e. micrograms, grams, kilograms of cells).

In addition, since the expression of NGN3 is necessary to initiate differentiation of endocrine cells, enhance the maturation of islet cells and maintain the function of islets, stimulation or induction of NGN3 expression in cells compromised with the Endocrine lineage (CHGA +) is an important stage to achieve in vitro differentiation from pluripotent stem cells to mature and functional p cells.

Summary of the invention

The invention is defined in the appended claims, with preferred and optional features set forth in the dependent claims. An in vitro method for producing a cell population that includes human pancreatic endodermal cells in human PDX1 is disclosed. The method may include contacting a cell population that includes cells of definitive endodermal lineage with a growth factor of the TGFp superfamily and a member of the Wnt family, thereby generating the cell population that includes PDX1 positive pancreatic endodermal cells. The TGFp superfamily growth factor can be selected from the group consisting of Nodal, Activina A, Activina B, BMP2, BMP4, GDF8, GDF-10, GDF-11 and GDF-15. The method can also include contacting the cell population that includes cells of definitive endodermal lineage with a Heregulin. The growth factor of the TGFp superfamily can be Activina A. The member of the Wnt family can be Wnt3a.

The cell population that includes PDX1 positive pancreatic endodermal cells can contact at least 25 ng / ml of the TGFp superfamily member. In embodiments, the cell population that includes PDX1-positive pancreatic endodermal cells is contacted with at least 50 ng / ml of the TGFp superfamily growth factor. In embodiments, the cell population that includes PDX1-positive pancreatic endodermal cells is contacted with at least 75 ng / ml of the TGFp superfamily member.

The cell population that includes PDX1 positive pancreatic endodermal cells can contact at least 25 ng / ml of the Wnt family member. The cell population that includes PDX1 positive pancreatic endodermal cells can contact at least 50 ng / ml of the Wnt family member. The cell population that includes PDX1 positive pancreatic endodermal cells can contact at least 75 ng / ml of the Wnt family member.

The cell population can contact 5-15 times less Heregulin than each of the TGFp superfamily members and the Wnt family member. At least about 50% of said cell population that includes PDX1-positive pancreatic endodermal cells may be PDX1-positive pancreatic endodermal cells. At least about 60% of the cell population that includes PDX1-positive pancreatic endodermal cells can be PDX1-positive pancreatic endodermal cells. At least about 70% of the cell population that includes PDX1-positive pancreatic endodermal cells can be PDX1-positive pancreatic endodermal cells. At least about 80% of the cell population that includes PDX1-positive pancreatic endodermal cells can be PDX1-positive pancreatic endodermal cells. At least about 90% of the cell population that includes PDX1-positive pancreatic endodermal cells can be PDX1-positive pancreatic endodermal cells.

PDX1-positive pancreatic endodermal cells may also express NKX6.1 and may not substantially express NGN3. PDX1 positive pancreatic endodermal cells do not need to express CHGA. PDX1 positive pancreatic endodermal cells can also express NKX6.1 and PFT1A and do not need to express substantially NGN3. PDX1-positive pancreatic endodermal cells can be multipotent cells that can differentiate into unipotent immature beta cells.

A culture of human pancreatic endodermal cells is disclosed that includes a definitive endodermal lineage cell, and a member of the TGFp superfamily and a member of the Wnt family. The TGFp superfamily member can be selected from the group consisting of Nodal, Activina A, Activina B, BMP2, BMP4, GDF8, GDF-10, GDF-11 and GDF-15. The method may also include contacting the cell population that includes cells of definitive endodermal lineage with a Heregulin. The TGFp superfamily member can be Activina A. The Wnt family member can be Wnt3a.

An unipotent human immature beta cell is revealed in vitro. The unipotent human immature beta cell can express INS and NKX6.1, and does not need to substantially express NGN3. The unipotent human immature beta cell may be able to differentiate into a mature beta cell. Differentiation can be in vivo. The unipotent human immature beta cell can be part of a cell population. At least 10% of the cell population can be immature beta cells. At least 20% of the cell population can be immature beta cells. At least 30% of the cell population can be immature beta cells. At least 40% of the cell population can be immature beta cells. At least 50% of the cell population can be immature beta cells. At least 60% of the cell population can be immature beta cells. At least 80% of the cell population can be immature beta cells. At least 90% of the cell population can be immature beta cells. At least 98% of the cell population can be immature beta cells. The human immature beta cell can only be hormonal. The unipotent human cell can express MAFB.

A method for producing an imipotent human immature beta cell is disclosed. The method may include contacting cells of the definitive human endodermal lineage in vitro with a member of the TGFp superfamily and a member of the Wnt family, thereby generating immature beta cells. The unipotent human immature beta cell can express INS, NKX6.1 and does not need to express substantially NGN3. The TGFp superfamily member can be selected from the group consisting of Nodal, Activina A, Activina B, BMP2, BMP4, GDf 8, GDF-10, GDF-11 and GDF-15. The method may also include contacting the cells of the definitive human endodermal lineage in vitro with a Heregulin. The TGFp superfamily member can be Activina A. The Wnt family member can be Wnt3a. The member of the TGFp superfamily can be BMP.

The method may further include contacting the cells of the definitive human endodermal lineage in vitro with nicotinamide, a retinoic acid or retinoic acid analogue, a rho kinase inhibitor or a gamma secretase inhibitor. Retinoic acid can be selected from the group consisting of all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA) and arotinoid acid (TTNPB). Retinoic acid can be all-trans retinoic acid (RA). The retinoic acid may be 13-cis-retinoic acid (13-cis-RA). The retinoic acid can be arotinoid acid (TTNPB). The Rho kinase inhibitor can be selected from the group consisting of Y-27632, Fasudil, H-1152P, Wf-536 and Y-30141. The rho kinase inhibitor can be Y-27632.

The gamma secretase inhibitor may be selected from the group consisting of gamma secretase I (GSI I) inhibitor, gamma secretase II (GSI II) inhibitor, gamma secretase III (GSI III) inhibitor, gamma inhibitor secretase IV (GSI IV), gamma secretase V (GSI V) inhibitor, gamma secretase VI (GSI VI) inhibitor, gamma secretase VII (GSI VII) inhibitor, gamma secretase IX (GSI IX) inhibitor , (DAPT), gamma secretase XI inhibitor (GSI XI), gamma secretase XII inhibitor, (GSI XII), gamma secretase XIII inhibitor (GSI XIII), gamma secretase XIV inhibitor (GSI XIV), gamma secretase XVI (GSI XVI) inhibitor, gamma secretase XVII (GSI XVII) inhibitor, gamma secretase XIX (GSI XIX) inhibitor, gamma secretase XX (GSI XX) inhibitor, gamma secretase XXI inhibitor (GSI XXI), gamma40 secretase I inhibitor, gamma40 secretase II inhibitor and RO4929097. The gamma secretase inhibitor may be RO4929097. The gamma secretase inhibitor may be GSI IV.

A method for producing mature beta cells in vivo is disclosed . The method may include, (a) contacting human cells of the definitive endodermal lineage in vitro with a member of the TGFp superfamily and a member of the Wnt family, thereby generating immature beta cells; (b) transplant the immature beta cells of step (a) into a mammalian subject; and allow immature beta cells to differentiate in vivo to produce a population of cells that includes mature beta cells. The method may also include allowing mature beta cells to produce insulin in response to glucose stimulation. The TGFp superfamily member can be selected from the group consisting of Nodal, Activina A and Activina B, GDF-8, GDF-11 and GDF-15. The method may also include contacting the cells of the definitive human endodermal lineage in vitro with a Heregulin. The TGFp superfamily member can be Activina A. The Wnt family member can be Wnt3a.

The cells of the definitive human endodermal lineage can contact at least 25 ng / ml of the TGFp superfamily member. The cells of the definitive human endodermal lineage may contact at least 5 ng / ml of the TGFp superfamily member. The cells of the definitive human endodermal lineage can contact at least 75 ng / ml of the TGFp superfamily member.

The cells of the definitive human endodermal lineage may contact at least 25 ng / ml of the Wnt family member. The cells of the definitive human endodermal lineage can contact at least 50 ng / ml of the Wnt family member. The cells of the definitive human endodermal lineage may contact at least 75 ng / ml of the Wnt family member.

The cells of the definitive human endodermal lineage may contact 5-15 times less Heregulin than the TGFp superfamily member and the Wnt family member. At least 10% of the cell population can be immature beta cells. At least 20% of the cell population can be immature beta cells. At least 30% of the cell population can be immature beta cells. At least 40% of the cell population can be immature beta cells. At least 50% of the cell population can be immature beta cells. At least 60% of the cell population can be immature beta cells. At least 70% of the cell population can be immature beta cells. At least 80% of the cell population can be immature beta cells. At least 90% of the cell population can be immature beta cells. Immature beta cells can express INS and NKX6.1, and do not need to express substantially NGN3. Immature beta cells can express INS, NKX6.1 and MAFB, and do not need to substantially express NGN3.

An unipotent human immature beta cell is revealed in vitro. The unipotent human immature beta cell can express INS and NKX6.1, and does not need to substantially express NGN3. The unipotent human immature beta cell may be able to mature and become a mature beta cell.

A method for producing mature beta cells in vivo is disclosed . The method includes: a. contacting cells of the definitive human endodermal lineage in vitro with a member of the TGFp superfamily and a member of the Wnt family, thereby generating immature beta cells; b. transplant the immature beta cells of step (a) into a mammalian subject; and c. allowing immature beta cells to mature in vivo to produce a population of cells that include insulin-secreting beta cells.

A unipotent human immature beta cell that expresses INS and NKX6.1 is disclosed, and that does not substantially express NGN3. The unipotent human immature beta cell may be able to mature and become a mature beta cell. The unipotent human immature beta cell can also express MAFB.

A unipotent human immature beta cell is revealed in vitro that expresses INS and NKX6.1, and that does not substantially express NGN3. The unipotent human immature beta cell may be able to mature and become a mature beta cell. The unipotent human immature beta cell can also express MAFB.

A method for producing an imipotent human immature beta cell is disclosed. The method includes contacting cells of the definitive human endodermal lineage in vitro with a member of the TGFp superfamily and a member of the Wnt family, thus producing an unipotent human immature beta cell. The immature beta cell Unipotent human can express INS, NKX6.1 and need not substantially express NGN3. The method may also include contacting the cells of the definitive human endodermal lineage in vitro with a Heregulin. The TGFp superfamily member can be Activina A. The Wnt family member can be Wnt3a.

A method for producing mature beta cells in vivo is disclosed . The method includes: a. contacting cells of the definitive human endodermal lineage in vitro with a member of the TGFp superfamily and a member of the Wnt family, thereby generating immature beta cells; b. transplant the immature beta cells of step (a) into a mammalian subject; and c. allowing immature beta cells to mature in vivo to produce a population of cells that include insulin-secreting beta cells. The method may also include contacting the cells of the definitive human endodermal lineage in vitro with a Heregulin. The TGFp superfamily member can be Activina A. The Wnt family member can be Wnt3a. The cells of the definitive human endodermal lineage may contact at least 50 ng / ml of the TGFp superfamily member. The cells of the definitive human endodermal lineage may contact at least 25 ng / ml of the Wnt family member. The cells of the definitive human endodermal lineage may contact 5-15 times less Heregulin than the TGFp superfamily member and the Wnt family member. Immature beta cells can express INS and NKX6.1, and do not need to express substantially NGN3. Immature beta cells can express iNs, NKX6.1 and MAFB, and do not need to express substantially NGN3.

A composition and method for differentiating pluripotent stem cells or precursor cells derived from pluripotent stem cells to endocrine and non-endocrine populations are disclosed. Endocrine cells can express at least CHGA (or CHGa), and non-endocrine cells do not need to express CHGA (or CHGA-). These cell populations may be referred to as endocrine (CHGA +) and non-endocrine (CHGA-) subpopulations, or simply CHGA + or CHGA- subpopulations, or simply endocrine and non-endocrine subpopulations. These endocrine and non-endocrine subpopulations may be multipotent progenitor / precursor subpopulations, such as non-endocrine multipotent pancreatic progenitor subpopulations or endocrine multipotent pancreatic progenitor subpopulations; or they may be unipotent subpopulations, such as immature endocrine cells, preferably immature beta cells, immature glucagon cells and the like.

A composition and method for differentiating pluripotent stem cells in vitro are revealed to pancreatic endodermal cell cultures (PEC) comprising both endocrine and non-endocrine subpopulations, in which NGN3 expression is suppressed. The expression of NKX2.2 can also be suppressed in the PEC culture. CHGA expression can also be suppressed in PEC culture. The expression of PDX1 and NKX6.1 can be expressed simultaneously in a single cell in the PEC culture. Less than 50%, preferably less than 40%, 30%, more preferably less than 20%, 10%, 5%, 2%, 1% of PEC culture cells can express NGN3, NKX2.2 or CHGA. The PEC culture can be enriched in the non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-). PEC culture can express markers (PDX1 and / or NKX6.1) for multipotent non-endocrine pancreatic progenitor subpopulation (CHGA-). PEC culture with abundant multipotent non-endocrine progenitor subpopulation (CHGA-) can be implanted in a mammalian host, maturing the graft and producing insulin in vivo in response to glucose. PEC culture with abundant multipotent non-endocrine pancreatic progenitor subpopulation (CHGA-) can mature in vivo at a faster rate than PEC cultures without an abundant non-endocrine multipotent progenitor subpopulation (CHGA-). The culture of PEC implanted with an abundant non-endocrine multipotent progenitor subpopulation (CHGA-) can have at least an improvement of twice in vivo function measured by the production of peptide C (insulin) in response to glucose stimulation approximately 10 weeks or approximately 12 weeks after implantation than implanted PEC cultures without a multipotent non-endocrine pancreatic progenitor subpopulation (CHGA).

A composition and a method for differentiating human pluripotent stem cells in vitro are disclosed , in which the expression of typical endocrine differentiation and development genes, such as NGN3 and NKX2.2, is suppressed until after the production of progenitor cells. non-endocrine multipotent pancreatic (simultaneous expression of CHGA and PDX1 / NKX6.1).

A method for differentiating pluripotent stem cells in vitro to PEC cultures is disclosed, comprising the steps of: (a) obtaining a population of endodermal cells from the upper digestive tract negative in PDX1; (b) contacting the cell population of step (a) with an agent that activates a member of the TGFp receptor family, thereby generating a cell culture comprising PEC, in which the PECs do not substantially express a selected marker of the group comprising CHGA, NGN3 or NKX2.2. PECs may comprise a subpopulation of cells that simultaneously express PDX1 and NKX6.1. PECs may comprise a subpopulation of non-endocrine multipotent pancreatic progenitor cells (CHGA-). The agent that activates a member of the TGFp receptor family can be selected from the group consisting of Nodal, Activina A, Activina B, BMP2, BMP4, GDF8, GDF-10, g Df -11, GDF-15 and a mixture of the same. Activin A can be added at 100 ng / ml, 75 ng / ml, 50 ng / ml, 25 ng / ml or 10 ng / ml in phase 3; and 15 ng / ml, 10 ng / ml or 5 ng / ml in phase 4. The culture of PEC formed by adding Activin A to a population of endodermal cells of the upper digestive tract negative in PDX1 may have at least one improvement. Twice the in vivo function measured by the production of C-peptide (insulin) in response to glucose stimulation than PEC cultures without an abundant non-endocrine multipotent progenitor subpopulation (CHGA-).

PEC culture with a multipotent non-endocrine pancreatic progenitor subpopulation (CHGA-) may have at least twice the improvement in function measured in vivo by insulin production in response to glucose stimulation than PEC cell cultures without subpopulations. multipotent non-endocrine progenitors (CHGA-) abundant.

A method for differentiating pluripotent stem cells in vitro is revealed to a cell population comprising PEC comprising the steps of: (a) obtaining a population of endodermal cells from the upper digestive tract negative in PDX1; (b) contacting the cellular population of step (a) with an agent that activates a member of the TGFp receptor family, thereby generating a population of p Ec, in which the PEC population does not substantially express a marker selected from the group comprising CHGA, NGN3 or NKX2.2. The PEC population may comprise cells that simultaneously express PDX1 and NKX6.1. The PEC population may comprise non-endocrine multipotent pancreatic progenitor cells (CHGA-). The agent that activates a member of the TGFp receptor family can be selected from the group consisting of Nodal, Activina A, Activina B, BMP2, BMP4, GDF8, GDF-10, GDF-11, GDF-15 and a mixture of same. The agent that activates a member of the TGFp receptor family can be Activin A. Activin A can be added at 100 ng / ml, 75 ng / ml, 50 ng / ml, 25 ng / ml or 10 ng / ml in phase 3; and 15 ng / ml, 10 ng / ml or 5 ng / ml in phase 4. The ligand of the EGF family can be selected from the group comprising Heregulin (also known as Neuregulin1 (NRG1)), Neuregulin2 (NRG2), Neuregulin3 (NRG3), Neuregulin4 (NRG4), EGF (epidermal growth factor) and betacellulin. Heregulin can be added at 2 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 15 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml , 70 ng / ml or 80 ng / ml. The culture of PEC formed by the addition of Activin A and Heregulin to a population of upper endodermal negative digestive cells in PDX1 can have at least an improvement of twice in vivo function measured by the production of C (insulin) peptide in Response to glucose stimulation than PEC cultures without abundant non-endocrine multipotent progenitor subpopulations (CHGA-).

A method for differentiating pluripotent stem cells in vitro to a cell culture comprising PEC is disclosed, comprising the steps of: (a) obtaining a population of endodermal cells from the upper digestive tract negative in PDX1; (b) contacting the cellular population of step (a) with at least one agent that activates a member of the TGFp receptor family, a ligand of the EGF family or an agent that activates a Wnt signaling pathway , alone or in combination, thus generating a cell culture comprising p Ec, in which the PECs do not substantially express a marker selected from the group comprising CHGA, NGN3 or NKX2.2. The PEC culture may comprise cells that simultaneously express PDX1 and NKX6.1. The PEC cell culture may comprise a multipotent non-endocrine pancreatic progenitor subpopulation (CHGA-). The agent that activates a member of the TGFp receptor family can be selected from the group consisting of Nodal, Activina A, Activina B, BMP2, BMP4, GDF8, GDF-10, g Df -11, GDF-15 and a mixture of the same. The agent that activates a member of the TGFp receptor family may be Activin A. An agent that activates a Wnt signaling pathway may be a member of the Wnt family selected from the group comprising WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16 and Wnt3A. The Wnt family member can be Wnt3A. Activin A can be added at 100 ng / ml, 75 ng / ml, 50 ng / ml, 25 ng / ml or 10 ng / ml in phase 3; and 15 ng / ml, 10 ng / ml or 5 ng / ml in phase 4. Heregulin can be added at 2 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 15 ng / ml , 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml or 80 ng / ml. WNT can be added at 5 ng / ml, 25 ng / ml, 50 ng / ml at 75 ng / ml, more preferably at 50 ng / ml.

Activin A can be added alone or in combination with WNT in phase 3. Activin A can be added with WNT or WNT and Heregulin in phase 3. Activin A can be added alone or in combination with Heregulin in phase 4.

More than 10%, preferably more than 20%, 30%, 40% and more preferably more than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% of the population cells Cellular may be the non-endocrine multipotent progenitor subpopulation (CHGA-). The culture of PEC cells formed by the addition of Activin A, Heregulin and WNT to a population of endodermal cells of the upper negative digestive tract in PDX1 can have at least an improvement of twice in vivo function measured by the production of insulin in response to glucose stimulation than PEC cell cultures without a multipotent non-endocrine progenitor subpopulation (CHGA-) abundant.

A composition and method for differentiating pluripotent stem cells in vitro to PEC cultures comprising minimal cells compromised with the endocrine lineage (CHGA +) are disclosed. Cells compromised with the endocrine lineage can be characterized by CHGA expression. Less than 50%, preferably less than 40%, 30%, more preferably less than 20%, 10%, 5%, 2%, 1% of the cells of the PEC culture may be cells compromised with the endocrine lineage (CHGA +) .

A composition and method for differentiating pluripotent stem cells in vitro are essentially disclosed to PEC cultures and to further differentiate the culture of PEC to endocrine cells in vitro. Endocrine cells can express CHGA. Endocrine cells can produce insulin in vitro. Endocrine insulin secreting cells in vitro can produce insulin in response to glucose stimulation. More than 10%, preferably more than 20%, 30%, 40% and more preferably more than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% of the population cells of cells can be endocrine cells.

A method for differentiating pluripotent stem cells in vitro to endocrine cells comprising the steps of: (a) obtaining a cell population comprising PEC is disclosed; (b) contacting the cell population of stage (a) with growth factors selected from the group consisting of a bone morphogenetic protein (BMP), a hedgehog protein inhibitor, platelet-derived growth factor (PDGF), member of the family of fibroblast growth factors, a retinoid, insulin growth factor 1 and 2 (IGF1 and IGF2) and nicotinamide, thus generating endocrine cells. Endocrine cells can express CHGA. Endocrine cells can be immature insulin producing beta cells in vitro. Endocrine cells can be loaded into an implantable semipermeable encapsulation device, mature and be able to produce insulin in response to glucose stimulation in vivo. More than 10%, preferably more than 20%, 30%, 40% and more preferably more than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% of the population cells Cell can be endocrine cells. SSH can be added at 10 g / ml to 100 ng / ml. PDGF can be added at 10 ng / ml to 100 ng / ml. BMP can be added at 10 g / ml to 20 ng / ml. FGF2 can be added at 2 ng / ml to 20 ng / ml. IGF1 or IGF2 can be added at 25 ng / ml to 100 ng / ml. The above compounds can be added in combination with each other. The member of the family of fibroblast growth factors can be FGF2. The retinoid can be retinoic acid and / or retinoic acid analogs, such as TTNPB or TT3, insulin-like growth factor I (IGF-I or IGF1) and -II (IGF-II or IGF2). The bone morphogenetic protein can be BMP4. The hedgehog protein inhibitor can be a sonic Hedgehog, KAAD-cyclopamine, KAAD-cyclopamine analogs, jervine, jervine analogs, antibodies that block the hedgehog pathway and any other hedgehog pathway function inhibitor known to the subject matter experts.

A method for differentiating pluripotent stem cells in vitro to endocrine cells comprising the steps of: (a) obtaining a cell population comprising PEC is disclosed; (b) contacting the cell population of step (a) with a gamma secretase inhibitor, thus forming endocrine cells in vitro. Endocrine cells can express CHGA. Endocrine cells can be immature insulin producing beta cells in vitro. Endocrine cells can be insulin secreting cells that produce insulin in response to in vitro glucose stimulation . Endocrine cells can be loaded into an implantable semipermeable encapsulation device, mature and be able to produce insulin in response to glucose stimulation in vivo. The gamma secretase inhibitor may be selected from the group comprising t-butyl ester of W- [A / - (3,5-difluropheacetyl-L-alanyl)] - S-phenylglycine (DAPT), RO44929097, 1- (S) -endo-N- (1,3,3) -Trimethylbicyclo [2.2.1] hept-2-yl) -4-fluorophenylsulfonamide, WPE-III31C, S-3- [W '- (3,5-difluorophenyl-alpha -hydroxyacetyl) -L-alanylyl] amino-2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepin-2-one, (A /) - [(S) -2-hydroxy- 3-methylbutyryl] -1- (L-alaninyl) - (S) -1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, BMS-708163 (Avagacestat ), BMS-708163, Semagacestat (LY450139), Semagacestat (LY450139), MK-0752, MK-0752, YO-01027, YO-01027 (Dibenzazepina, DBZ), LY-411575, LY-411575 or LY2811376. The gamma secretase inhibitor may be present in the cell culture or in the cell population at a concentration ranging from about 0.01 pM to about 1,000 pM. The gamma secretase inhibitor may be present in the cell culture or in the cell population at a concentration ranging from about 0.1 pM to about 100 pM. The gamma secretase inhibitor can be removed after its addition.

Compositions and methods for differentiating human pluripotent stem cells in vitro to endocrine cells are disclosed. Endocrine cells can express CHGA. Endocrine cells can produce insulin in vitro. Endocrine cells can be immature endocrine cells, such as immature beta cells. Insulin producing cells in vitro can produce insulin in response to glucose stimulation.

A method for producing insulin in vivo in a mammal is disclosed, said method comprising: (a) loading a population of endocrine cells into an implantable semipermeable device; (b) implant the device with the endocrine cell in a mammalian host; and (c) mature the population of endocrine cells in said device in vivo, in which at least some of the endocrine cells are insulin secreting cells that produce insulin in response to glucose stimulation in vivo, thereby producing insulin in vivo to the mammal The endocrine cell may be derived from a cellular composition comprising PEC with a higher multipotent non-endocrine pancreatic progenitor subpopulation (CHGA-). The endocrine cell can be derived from a cellular composition comprising PEC with a reduced endocrine subpopulation (CHGA +). The endocrine cell may be an immature endocrine cell, preferably, an immature beta cell.

Endocrine cells created in vitro from pluripotent stem cells can express more PDX1 and NKX6.1 compared to pancreatic endodermal populations positive in PDX-1, or non-endocrine subpopulations (CHGA-) that are positive in PDX1 / NKX6 .one. Endocrine cells created in vitro from pluripotent stem cells can express PDX1 and NKX6.1 relatively more than the non-endocrine multipotent pancreatic progenitor subpopulation of PEC (CHGA-). A bone morphogenic protein (BMP) and a retinoic acid analog (RA) alone or in combination can be added to the cell culture to obtain endocrine cells with a higher expression of PDX1 and NKX6.1 compared to the multipotent non-endocrine progenitor subpopulation of PEC (CHGA-). BMP can be selected from the group comprising BMP2, BMP5, BMP6, BMP7, BMP8 and BMP4, and more preferably BMP4. The retinoic acid analog can be selected from the group comprising all trans retinoic acid and TTNPB (Arotinoid acid 4 - [(£) -2- (5,6,7,8-tetrahydro-5,5,8,8 -tetramethyl-2 naphthalenyl) -1-propenyl] benzoic acid, or AM-580 0.1-10 pM (4 - [(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2 acid -naphthalenyl (carboxamido] benzoic) and more preferably TTNPB.

A method for differentiating pluripotent stem cells in vitro to immature endocrine and endocrine cells, preferably immature beta cells, is disclosed, comprising dissociating and associating the aggregates again. Dissociation and reassociation can occur in phase 1, phase 2, phase 3, phase 4, phase 5, phase 6 or phase 7, or combinations thereof. The final endodermal, endodermic cells of the upper digestive tract negative in PDX1, endodermal cells of the upper digestive tract positive in PDX1, PEC and / or endocrine and endocrine progenitors / precursors can be dissociated and re-associated. Dissociated and re-aggregated cell aggregates from phase 7 may consist of less non-endocrine subpopulations (CHGA-) compared to endocrine subpopulations (CHGA +). More than 10%, preferably more than 20%, 30%, 40% and more preferably more than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% of the population cells Cellular can be endocrine cells (CHGA +).

A method for differentiating in vitro pluripotent stem cells to endocrine cells is revealed by eliminating the endocrine cells produced during the production of PEC from phase 4, thus enriching in non-endocrine pancreatic progenitor subpopulation (CHGA-) which is PDX1 + and NKX6. 1+.

A method for differentiating pluripotent stem cells in vitro to endocrine cells is disclosed, in which phase 6 and phase 7 cells, as defined in Table 17, are grown at low Matrigel levels of about 0.02% at 2%, preferably from about 0.02% to 0.05%, preferably from about 0.05% to 1%, preferably 0.05%.

A method for improving cell adhesion of PEC and endocrine cell aggregates is disclosed by treating cell cultures with Matrigel and a rho-kinase or ROCK inhibitor. The rho kinase or ROCK inhibitor can be Y-27632.

Cultures of PECs enriched in the multipotent non-endocrine progenitor subpopulation (CHGA-) can be prepared without adding a member of the nogina family in phase 3 and / or phase 4. PEC cultures that are relatively packed with cells Engaged with the endocrine lineage (CHGA +) can be prepared without adding a member of the nogina family in phase 3 and / or phase 4. The member of the nogina family can be a compound selected from the group comprising nogina , choline, Folistatin, Folistatin-like proteins, Cerberus, Coconut, Dan, Gremlina, Sclerostin, PRDC (protein related to Dan and Cerberus).

A method for maintaining endocrine cells in culture is disclosed by culturing them in a medium comprising high exogenous glucose levels, in which the exogenous glucose added is about 1 mM to 25 mM, about 1 mM to 20 mM, about 5 mM at 15 mM, from about 5 mM to 10 mM, from about 5 mM to 8 mM. The media can be a DMEM-based, CMRL-based or RPMI-based medium.

A method for differentiating pluripotent stem cells in vitro to endocrine cells with and without dissociating and re-associating cell aggregates is disclosed. Non-dissociated or dissociated and re-associated cell aggregates can be cryopreserved or frozen in phase 6 and / or phase 7 without affecting the in vivo function of endocrine cells. Cryopreserved endocrine cell cultures can be thawed, cultured and, when transplanted, work in vivo.

A culture system for differentiating pluripotent stem cells from endocrine cells is disclosed, the culture system comprising at least one agent capable of suppressing or inhibiting endocrine gene expression during the first stages of differentiation and an agent capable of inducing endocrine gene expression during later phases of differentiation. An agent capable of suppressing or inhibiting endocrine gene expression can be added to the culture system consisting of cells in the upper pancreatic digestive tract in PDX1. An agent capable of inducing endocrine gene expression can be added to the culture system consisting of pancreatic endodermal progenitor cells PDX1 or PEC positive. An agent capable of suppressing or inhibiting endocrine gene expression can be an agent that activates a family of TGFbeta receptors, preferably it is Activin, preferably it is high levels of Activin, followed by low levels of Activin. An agent capable of inducing endocrine gene expression may be a gamma secretase inhibitor selected from a group consisting of N- [N- (3,5-diflurophenyl-L-alanyl) t-butyl ester] - S-phenylglycine (DAPT), RO44929097, DAPT (N- [N- (3,5-difluoropheacetyl-L-alanyl)] -S-phenylglycine t-butyl ester, 1- (S) -endo-N- (1,3 , 3) -trimethylbicyclo [2.2.1] hept-2-yl) -4-fluorophenylsulfonamide, WPE-III31C, S-3- [N '- (3,5-difluorophenyl-alpha-hydroxyacetyl) -L-alanylyl] amino -2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepin-2-one, (N - [((S) -2-hydroxy-3-methyl-butyryl] -1- ( L-alaninyl) - (S) -1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, BMS-708163 (Avagacestat), BMS-708163, Semagacestat ( LY450139), Semagacestat (LY450139), MK-0752, MK-0752, YO-01027, YO-01027 (Dibenzazepine, DBZ), LY-411575, LY-411575 or LY2811376. High levels of Activin mean levels above 40 ng / ml, 50 ng / ml and 75 ng / ml High levels of Activin can be used during phase 3 or before production ion of pancreatic endodermal cells of the upper digestive tract. Low levels of Activin mean less than 30 ng / ml, 20 ng / ml, 10 ng / ml and 5 ng / ml. Low levels of Activin can be used during phase 4 or for the production of PEC. The endocrine gene that is inhibited or induced can be NGN3. Activin A and Wnt3A can be used alone or in combination to inhibit endocrine expression, preferably, to inhibit NGN3 expression before the production of pancreatic endodermal cells of the upper digestive tract, or preferably during phase 3. A gamma-secretase inhibitor, preferably RO44929097 or DAPT, in the culture system to induce endocrine gene expression after PEC production, or preferably during phases 5, 6 and / or 7.

An in vitro cell culture comprising endocrine cells is disclosed in which at least 5% of said human cells express an endocrine marker selected from the group consisting of insulin (INS), homeotic box 1 of NK6 (NKX6.1), box pancreatic and duodenal homeotic 1 (PDX1), locus related to transcription factor 2 (NKX2.2), paired box 4 (PAX4), neurogenic differentiation 1 (NEUROD), Forkhead box A1 (FOXA1), Forkhead box A2 (FOXA2) , zinc finger of the snail family 2 (SNAIL2) and family A and B of musculoaponeurotic fibrosarcoma oncogenes (MAFA and MAFB), and does not essentially express a marker selected from the group consisting of neurogenin 3 (NGN3), islet 1 ( ISL1), hepatocyte nuclear factor 6 (HNF6), GATA 4 binding protein (GATA4), GATA 6 binding protein (GATA6), specific transcription factor of pancreas 1a (PTF1A) and SRY (sex determining region Y ) -9 (SOX9), in which said endocrine cells are unipoten tes and can ripen pancreatic beta cells.

Endocrine cells can be immature beta cells.

At least 10% of said human cells may be immature beta cells.

At least 20% of said human cells may be immature beta cells.

At least 50% of said human cells can be immature beta cells.

The cells can be non-recombinant.

The cells can be derived from human pluripotent stem cells selected from a group consisting of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC), partenote cells, embryonic carcinoma cells (EC), mesedermal cells, definitive endodermal cells , pancreatic endodermal cells of the upper digestive tract negative in PDX1-1, pancreatic endodermal cells of the upper digestive tract positive in PDX1 and endodermal pancreatic cells (PEC).

A method for producing an immature beta cell is disclosed, said method comprising the steps of: a) contacting a population of endogenous upper digestive tract cells in PDX1 with a factor that activates a member of the TGFp receptor family, thus producing a population of positive pancreatic endodermal cells in PDX1; and b) contacting the population of positive pancreatic endodermal cells in PDX with a gamma secretase inhibitor, thus producing an immature beta cell expressing INS and NKX6.1. The population of upper endodermal cells of the negative digestive tract in PDX1 can be contacted. Contact with a member of the Wnt family and the Heregulins.

The member of the TGFbeta receptor family can be selected from a group consisting of Activina A, Activina B, Nodal, GDF-8, GDF-10, GDF-11 and GDF15.

The member of the TGFbeta receptor family can be Actin A, GDF-8 or GDF-11.

The member of the TGFbeta receptor family may be Activina A.

The Wnt family member can be selected from a group consisting of WNT 1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9B, WNT9B, WNT9B, WNT9B, WNT9B, WNT9B, WNT9B, WNT9B, WNT9B, WNT9B, WNT9B WNT11, WNT16 and WNT3A.

The Wnt family member can be Wnt3a.

The member of the Heregulin family (HRG) can be HRG1, HRG 2, HRG 3 and HRG4.

The member of the HRG family can be HRG1 and HRG2.

A method for producing insulin in vivo is disclosed, said method comprising the steps of: a) contacting a population of endogenous upper digestive tract cells in PDX1 with a factor that activates a member of the TGFp receptor family, producing thus a population of positive pancreatic endodermal cells in PDX1; and b) contacting the population of PDX positive pancreatic endodermal cells with a gamma secretase inhibitor, thus producing immature beta cells expressing INS and NKX6.1; c) implant the immature beta cells of step (b); and d) mature the cells to mature beta cells that secrete insulin in vivo in response to glucose stimulation.

In phase 7, agents can still modulate gene expression. A member of the FGF superfamily can be added in phase 7. FGF2 can be added in phase 7. FGF2 can increase the expression of SST and / or suppress the expression of INS, GCG or GHRL. BMP can be added in phase 7. BMP can increase the expression of INS, PDX1 or IDI.

A population of immature beta cells is revealed that gives rise to function in vivo. The immature beta population can be CHGA +. The population of immature beta cells may be able to function in vivo, that is, when transplanted, insulin is secreted in response to blood glucose. The endocrine cell population, when transplanted, can develop and mature to become functional cells of the pancreatic islets. The population of immature beta cells can be enriched in endocrine cells (or depleted of non-endocrine cells). More than 50% of the cells of the immature beta cell population can be CHGA +. More than 60% or 70% or 80% or 90% or 95% or 98% or 100% of the cells of the immature beta cell population may be CHGA-. Less than about 50% of the cells in the immature beta cell population can be CHGA-. Less than about 15% of the cells of the immature beta cell population can be CHGA-. Less than about 10% or 5% or 3% or 2% or 1% or 0.5% or 0% of the cells of the immature beta cell population may be CHGA-. In addition, the expression of certain markers can be suppressed during the production of immature beta cells, such as the expression of NGN3 during phase 3. The population of immature beta cells may be only hormonal (eg, INS only). The immature beta cell population can jointly express other cell markers, including NKX6.1 and PDX1. The population of immature beta cells can both express the hormone individually and jointly express other cell markers, including NKX6.1 and PDX1. The immature beta cell population may have more INS cells that express the hormone individually as a percentage of the total INS population. The population of immature beta cells can have at least 50% of INS cells that express the hormone individually as a percentage of the total INS population. The population of immature beta cells can be CHGA + / INS / NKX6.1+ (triple positive). More than 25% of the cells of the immature beta cell population can be CHGA + / INS + / NKX6.1 + (triple positive). More than 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95% or 100% of the cells of the immature beta cell population can be CHGA + / INS + / NKX6.1 + (triple positive).

A population of immature enriched beta cells formed by dissociation and reintegration of cell aggregates is disclosed. The population of immature enriched beta cells can be dissociated and added back in phase 7. The population of immature beta cells can be dissociated and added again at approximately day 25, day 26, day 27 or day 28 or day 28 29 or 30 or more.

Complex means are important for the maintenance of endocrine cells. Complex media can be CMRL or RPMI. Complex media can be used in phase 6 or phase 7, or both in phase 6 and in phase 7. CMRL increased endocrine marker expression. RPMI increased endocrine marker expression.

Cryopreservation does not reduce immature beta cell populations in phase 7. Cryopreservation does not reduce CHGA + expression in phase 7.

Phase 7 cells are positive in NKX6.1. The cells of phase 7 are positive in peptide C. The cells of phase 7 are positive in both NKX6.1 and peptide C. The cells of phase 7 are of a single hormone. Phase 7 cells individually express INS and GCG.

Dissociation and reintegration of cells in phase 7 does not affect function in vivo.

Cryopreservation of cells in phase 7 does not affect function in vivo.

Phase 7 cells can be cryopreserved and retain their function in vivo.

Increasing the concentration of glucose increases the expression of INS, GCG or SST. It is preferred that the glucose concentration be increased in phase 6 or in phase 7, or both. It is preferred that GHRL expression decrease with increasing glucose concentration.

An immature beta cell is revealed in vitro that is a single hormone (INS only).

An immature beta cell is revealed in vitro that jointly expresses NKX6.1 and PDX1.

An immature beta cell is revealed in vitro of a single hormone (INS only) and jointly expresses NKX6.1 and PDX1.

An immature beta cell is revealed in vitro that is a single hormone (INS only), which jointly expresses NKX6.1 and PDX1, and can mature in vivo to a cell that can secrete insulin in response to blood glucose.

An immature beta cell is revealed in vitro that can mature in vivo to a cell that can secrete insulin in response to blood glucose.

An immature beta cell is revealed in vitro which is CHGA +, INS + and NKX6.1. More than 10% of the cell population In vitro total can comprise immature beta cells that are CHGA +, INS + and NKX6.1 +. It is immature beta if more than 50% of the total in vitro cell population comprises immature beta cells that are CHGA +, INS + and NKX6.1 +. It is immature beta if more than 80% of the total in vitro cell population comprises immature beta cells that are CHGA +, INS + and NKX6.1 +.

Unripe purified beta cells are revealed. Purified immature beta cell precursors are disclosed. Immature beta cells or beta cell precursors can be purified from phase 7 cell populations using a zinc sensor. Dark fluorescence due to the presence of a Py 1 sensor attached to zinc can be enriched in beta cells. The bright fluorescence due to the presence of a Py 1 sensor attached to zinc can be enriched in alpha cells. A method for purifying beta cells using a zinc sensor is disclosed. A method for purifying alpha cells using a zinc sensor is disclosed. Purified alpha cells are disclosed. Purified alpha cell precursors are disclosed.

An enriched cell population is revealed that expresses markers of the beta cell lineage, such as INS, IAPP, PDX1, NKX6.1, PAX4, PCSK1, G6PC2, GCK or SLC30A8.

An enriched cell population is revealed that expresses markers of the alpha cell lineage, such as GCG, ARX or SLC30A8.

An enriched cell population is revealed, in which more than 50% of the cells are INS positive. The cell population can have more than 90% positive INS cells. From 50% to 99% of the cells can be INS positive cells.

A method for producing insulin in vivo is disclosed, comprising: a) obtaining a population of CHGA + cells in vitro ; and b) transplant CHGA + cells producing insulin in vivo.

CHGA + cells can dissociate and regrow in phase 7.

CHGA + cells can express PDX1 and NKX6.1

CHGA + cells can express INS and NKX6.1.

CHGA + cells can be cultured in CMRL or RPMI.

CHGA + cells can be cryopreserved and thawed before transplantation.

CHGA + cells can be cultured in media containing a high glucose content.

An endocrine cell is revealed in vitro that can result in a physiologically functional insulin secretory cell in vivo.

An appropriately specified immature beta cell is revealed in vitro that can result in a physiologically functional insulin secretory cell in vivo.

Properly specified immature beta cells can dissociate and regrow in phase 7.

Properly specified immature beta cells can express PDX1 and NKX6.1

Properly specified immature beta cells can express INS and NKX6.1.

Properly specified immature beta cells can be cultured in CMRL or RPMI.

Properly specified immature beta cells can be cryopreserved and thawed before transplantation.

Properly specified immature beta cells can be cultured in media containing a high glucose content.

A method for producing a population of immature enriched beta cells in vitro is disclosed, comprising: obtaining a population of CHGA + cells and classifying it. A method for producing an enriched cell population that expresses INS, IAPP, PDX1, NKX6.1 or PAX4 in vitro is disclosed, comprising: obtaining a population of CHGA + cells and classifying it.

A method for producing a population of enriched alpha cells in vitro is disclosed, comprising: obtaining a population of CHGA + cells and classifying it.

A method for producing an enriched cell population that expresses GCG or ARX in vitro is disclosed, comprising: obtaining a population of CHGA + cells and classifying it.

Enriched populations of alpha or beta cells can be classified using a Py 1 zinc sensor.

Immature beta cells can express INS and NKX6.1, and do not need to express substantially NGN3. Immature beta cells can express INS, NKX6.1 and MAFB, and do not need to substantially express NGN3.

Brief description of the drawings

FIG. 1 is a photographic image of a suspension culture of dedifferentiated reprogrammed cell aggregates, or also referred to herein, iPS cells. See Example 1.

FIG. 2A-L are bar graphs showing relative levels of OCT4 gene expression (FIG.2A), BRAQUIURIA (FIG.2B), CER1 (FIG.2C), GSC (FIG.2D), FOXA2 (FIG.2E) , FOXA1 (FIG.2F), HNF6 (FIG.2G), PDX1 (FIG.2H), PTF1A (FIG.2I), NKX6.1 (FIG.2J), NGN3 (FIG.2K) and INS (FIG.2L ). Expression levels are normalized with respect to the average expression levels of the constitutive genes, cyclophilin G and TATA binding protein expression (TBP). The graphs represent the positive regulation at the lowest data point in the data set. See Example 1.

FIG. 3 are immunocytochemical (ICC) photomicrographs of cultures of human iPS cells (E2021 using hIPS G4 cell line) of phase 4 differentiation (PDX1-positive pancreatic endodermal cells) using PDX-1-specific antibodies (panel A); NKX6.1 (panel B); PTF1A (panel C); and Dapi (panel D). See Example 2.

FIG. 4 are immunocytochemical (ICC) images of iPS cell cultures (E2021 using the G4 hIPS cell line) of phase 5 differentiation using glucagon specific ligands (panel A); insulin (panel B); Somatostatin (panel C); and Dapi (panel D). See Example 2.

FIG. 5A-B is a matrix location map and the matrix legend provided in the phosph-RTK Proteome Profiler ™ human antibody matrices of R&D Systems. The location map of FIG. 5A shows the coordinates or location of the RTK antibodies. The identity or name of the RTK family and the antibodies are described in the legend, FIG. 5B. The positive signals observed in the revealed film, therefore, can be identified by superimposing a transparency as in FIG. 5A and identifying the signals in reference to the overlap coordinates (FIG. 5A) with the name of the RTK of FIG. 5B. See Example 4.

FIG. 6 are an analysis of RTK matrices of pancreatic endodermal cells derived from iPS cells (PEC) under four different conditions (panels A, B, C and D described in Example 5. Tyrosine phosphorylation of certain RTKs is observed by identification of high to low intensity signals Family members IGF1R / IR and ERBB (EGFR) are identified or framed See Example 5.

FIG. 7A-C are graphs showing the concentrations of human C-peptide and insulin in sera of implanted mice for experiments E2314, E2356 and E2380 (FIG. 7A), E2347 (FIG. 7B) and E2354 (FIG. 7C) . FIG. 7A: Treatment of cells expressing PDX1 with heregulin in vitro improves insulin secretion stimulated with glucose after transplantation and maturation in vivo. FIG. 7B: Glucose-stimulated C-peptide levels at 23 weeks after transplantation, 3 weeks before STZ induction of the mouse diabetes model. Mice implanted with PEC were analyzed at the times indicated after grafting to determine fasting serum human C-peptide levels, and 30 min and 60 min after intraperitoneal glucose administration. In FIG. 7C, PEC was encapsulated with cell encapsulation devices (Encaptra® EN20 or EN20, ViaCyte, San Diego, CA) and, in some cases, the devices had microperforations (pEN20, ViaCyte, San Diego, CA). Such devices have been described in US Pat. No. 8,278,106. See Example 7.

FIG. 8A-B are graphs showing the results of blood glucose tests of mice treated with STZ for Experiment No. 2347. Glucose-sensitive insulin secreting beta cells derived from blood glucose control are shown. iPEC implanted in the STZ-induced diabetes model. FIG. 8A: shows blood glucose for each of the 13 mice (baseline with and without Heregulin). Figure 8B shows the combined mean measurements for each treatment (baseline with and without Heregulin). Measurements of non-fasting blood glucose levels are shown for the 13 mice implanted with iPEC grafts up to 14 days before being treated with STZ (day 0), and for the same mice after STZ treatment and after explanted the grafts. Animals treated with STZ received STZ approximately 26 weeks after graft transplantation (day 0). At 28 weeks after the graft transplant, approximately 2 weeks after the start of the STZ treatment, the iPEC grafts were explanted (removed). Non-fasting blood glucose measurements were collected over time for each animal. See Example 7.

FIG. 9A-E are bar graphs showing the relative levels of gene expression of PDX1 (FIG. 9A), NKX6.1 (FIG. 9B), PTF1A (FIG. 9C), NKX2.2 (FIG. 9D) and NGN3 ( FIG. 9E). See Example 8.

FIG. 10 is a photographic image of a suspension culture of aggregates in which the endodermal cells of the upper digestive tract negative in PDX1 (phase 2) were differentiated to endodermal cells of the upper digestive tract positive in PDX1 (phase 3) with the addition of 50 ng / ml of Activina A. See Example 8.

FIG. 11 is a photographic image of suspension cultures of aggregates at the end of phase 3 (day 8, upper panel) and 4 (day 12, lower panel) using 5 ng / ml (ACT5) or 10 ng / ml (ACT10) of Activin A in phase 3; or 5 ng / ml (ACT5) of Activin in phases 3 and 4. See Example 8.

FIG. 12 are photographic images of cultures in suspension during phase 4 (AE, upper panel) or at the end of phase 4 (AE, lower panel). The top panel shows the addition of 10 ng / ml of Activin A and 2 ng / ml of Heregulin (ACT 10 HGN 2) or 20 ng / ml of Activin A and 2 ng / ml of Heregulin (ACT 20 HGN 2), or 20 ng / ml of Activin A and 10 ng / ml of Heregulin (ACT 20 HGN 10) or 10 ng / ml of Activin A, 2 ng / ml of Heregulin and 50 ng / ml of WNT (ACT 10 HGN 2 WNT 50) in phase 3. The bottom panel shows the addition of 25 ng / ml of Activina A (ACT 25), 50 ng / ml of Activina A (ACT 50), 75 ng / ml of Activina A (ACT 75) or 100 ng / ml of Activin A (ACT 100) in phase 3. All conditions received a low level of Activin and Heregulin in phase 4. See Example 9.

FIG. 13A-C are bar graphs showing the relative levels of gene expression of NGN3 (FIG. 13A), NKX2.2 (FIG. 13B) and NKX6.1 (FIG. 13C) when Activin, Heregulin and WNT (AHW) were added ) in phase 3, and when Activin and Heregulin (AH) were added in phase 4 as described in Example 9.

FIG. 14A-B are bar graphs showing the relative levels of gene expression of NKX6.1 (FIG. 14A) and NGN3 (FIG. 14B) when Activin, Heregulin and WNT (AHW) are added in phase 3, and when added Activin and Heregulin (AH) in phase 4 as described in Example 10.

FIG. 15 is a graph showing the concentrations of human C-peptide in sera from mice implanted with encapsulated PEC (control) and encapsulated modified PEC (produced using Activin, Heregulin and WNT (AHW) in phase 3, and Activin and Heregulin (AH) in phase 4). Expression levels were analyzed 11 weeks after fasting, 30 min and 60 min after intraperitoneal glucose administration. See Example 10.

FIG. 16 is a graph showing the addition of human C-peptide concentrations in sera from mice implanted with encapsulated PEC (control) and encapsulated modified PEC (produced using Activin, Heregulin and WNT (AHW) in phase 3, and Activin and Heregulin (AH) in phase 4). Expression levels were analyzed 14 weeks after fasting, 30 min and 60 min after intraperitoneal glucose administration. See Example 10.

FIG. 17 is a bar graph showing the relative gene expression levels of Neurogenin 3 on days 13, 15 and 17 after treatment with a gamma secretase inhibitor in phase 5 as described in Example 11.

FIG. 18A-D are bar graphs showing the relative levels of gene expression of PDX1 (FIG. 18A), NKX6.1 (FIG. 18B), SOX9 (FIG. 18C) and PTF1A (FIG. 18D) after differentiation according with Table 17 for phases 1-5, for phases 6 and 7, only FBS, Matrigel and a rho-kinase inhibitor were used in addition to the base DMEM medium as described in Example 12.

FIG. 19A-D are bar graphs showing the relative levels of gene expression of INS (FIG. 19A), GCG (FIG. 19B), GHRL (FIG. 19C) and SST (FIG. 19D) under the same differentiation conditions as in FIG.

18 and as described in Example 12.

FIG. 20 is a graph showing the concentrations of human C-peptide in sera from mice implanted with encapsulated endocrine cells, as described in Example 12 (E2395, Rag 2, week 10 GSIS). Expression levels were analyzed 10 weeks after fasting grafting and 60 min after intraperitoneal glucose administration. See Example 12.

FIG. 21 is a graph showing the concentrations of human C-peptide in sera from mice implanted with encapsulated endocrine cells, as described in Example 12 (E2395, Rag 2, week 15 GSIS). Expression levels were analyzed 15 weeks after fasting grafting and 60 min after intraperitoneal glucose administration. See Example 12.

FIG. 22A-D are photographic micrographs showing aggregates of differentiated and matured endocrine cells in vivo as described in Example 12. The cell field is stained with DAPI (FIG. 22A) or antibodies against NKX6.1 (FIG. 22B and FIG. 22C, nuclear staining), and chromogranin A (FIG. 22B and FIG. 22D, cytoplasmic). The same field is shown in all images. See Example 12.

FIG. 23A-C are photographic micrographs showing aggregates of endocrine cells as described in Example 12. FIG. 23A shows a field of cells stained for INS (cytoplasmic) and NKX6.1 (nuclear). FIG. 23B shows the same field of cells stained for INS (cytoplasmic) and PDX1 (nuclear). FIG. 23C shows the same field of cells stained with DAPI. See Example 12.

FIG. 24A-D are bar graphs showing Nanostring mRNA data of the relative gene expression levels of INS (FIG. 24A), NKX6.1 (FIG. 24B), PDX1 (FIG. 24C) and ID1 (FIG. 24D) under the differentiation conditions described in Example 13.

FIG. 25A-C are photographic micrographs showing aggregates of endocrine cells as described in Example 13. FIG. 25A is a control without the addition of TT or BMP, FIG. 25B shows the addition of TT in phase 7, FIG. 25C shows the addition of BMP in phase 7. FIG. 25A-C show staining for peptide C (cytoplasmic) and PDX1 (nuclear).

FIG. 26A-B are photographic micrographs showing aggregates of endocrine cells as described in Example 13. FIG. 26A is treated with TT and BMP in steps 6 and 7, and FIG. 26B shows the addition of TT and BMP in phase 7. FIG. 26A and B show staining for peptide C (cytoplasmic) and PDX1 (nuclear).

FIG. 27A-C are photographic micrographs showing aggregates of endocrine cells as described in Example 13 and represent the same fields as in FIG. 25. FIG. 27A is a control without the addition of TT or BMP, FIG. 27B shows the addition of TT in phase 7, FIG. 27C shows the addition of BMP in phase 7. FIG. 27A-C show staining for peptide C (cytoplasmic) and NKX6.1 (nuclear).

FIG. 28A-B are photographic micrographs showing aggregates of endocrine cells as described in Example 13 and represent the same fields as in FIG. 26. FIG. 28A is treated with TT and BMP in phases 6 and 7, and FIG. 28B shows the addition of Tt and BMP in phase 7. FIG. 28A and B show staining for peptide C (cytoplasmic) and NKX6.1 (nuclear).

FIG. 29A-E are bar graphs showing relative levels of gene expression of endocrine aggregates treated with (right bar; d21 g13d-d15) or untreated (right bar; d21 g13d13-d15 + IAS d15) nicotinamide in phase 6 and analyzed on day 21 as described in Example 13.

FIG. 30 shows photographic images (days 21, 22 and 23) of suspended cultures of aggregates of dissociated and regrouped endocrine cells at the beginning of phase 7, followed by the addition of FBS alone, both FBS (panel A) and Matrigel at 0, 05% (MG; panel B), both FBS and Y-27632 10 pM (Y; panel C) or all three, FBS, Matrigel (Mg) 0.05% and Y-27632 10 pM (Y; panel D) as described in Example 14.

FIG. 31A-D are bar graphs showing the relative levels of gene expression of NKX6.1 (FIG. 31A), NKX2.2 (FIG. 31B), PDX1 (FIG. 31C) and INS (FIG. 31D) that compare the Differentiation conditions with and without Nogina for 1, 2 and 3 days. See Example 15.

FIG. 32A-C are bar graphs showing Nanostring mRNA data of the relative gene expression levels of INS and GCG (FIG. 32A), GHRL and SST (FIG. 32B) and PDX1 and ID1 (FIG. 32C) that describe the effect on gene expression by the indicated factors. See Example 16.

FIG. 33A-C are bar graphs showing Nanostring mRNA data of the relative gene expression levels of PDX1, NKX6.1, PTF1A, SOX9 (FIG. 33A); INS, GCG, PPY, GHRL (FIG. 33B) and PCSK1, GCK, SLC30A8, G6PC2 (FIG. 33C) describing endocrine subpopulations (CHGA +) and non-endocrine subpopulations (CHGA-) in regrouped crops compared to crops not regrouped See Example 17.

FIG. 34 is a graph showing the concentrations of human C-peptide in sera from mice implanted with encapsulated endocrine cells, as described in Example 18, where endocrine cell types (CHGA +) were enriched by dissociation and reintegration at baseline. of phase 7 (re-aggregated sample) and compared to differentiated cells in the same way but not re-aggregated (control sample). Expression levels were analyzed 21 weeks after fasting, 30 min and 60 min after intraperitoneal glucose administration. See Example 18.

FIG. 35A-C are bar graphs showing Nanostring mRNA data of the relative gene expression levels of CHGA, INS, GCG, GHRL, PPY, SST (FIG. 35A); GCK, G6CP2, UCN3, PCSK1 (FIG. 35B); HNF1a, HNF4A, SLC30A8, CADH1 (FIG. 35C) describing the effect of DMEM and CMRL based media during phase 7. See Example 19.

FIG. 36A-C are photographic micrographs showing the aggregates of endocrine cells of phase 7 as described in Example 20. The aggregates have not been added again in phase 7. The same field in FIG.

36A and in FIG. 36B are stained for INS (cytoplasmic) and GCG (cytoplasmic), respectively; and FIG. 36C is stains for NKX6.1 (nuclear) and C-peptide (cytoplasmic).

FIG. 37A-C are photographic micrographs showing the aggregates of endocrine cells of phase 7 as described in Example 20. The aggregates have been added again in phase 7. The same field in FIG. 37A and FIG. 37B are stained for INS (cytoplasmic) and GCG (cytoplasmic), respectively; and FIG. 37C is stained for NKX6.1 (nuclear) and C-peptide (cytoplasmic).

FIG. 38 is a graph showing the concentrations of human C-peptide in sera from mice implanted with encapsulated endocrine cells, as described in Example 20, whereby the cell cultures were frozen (cryopreserved) and thawed (F / T), and did not dissociate or reintegrate (left side), or did not freeze (freshly prepared) and dissociate and regregate at the beginning of phase 7 (right side). Expression levels were analyzed 12 weeks after fasting, 30 min and 60 min after intraperitoneal glucose administration. See Example 21.

FIG. 39A-B are bar graphs showing Nanostring mRNA data of the relative gene expression levels of INS, GCG (FIG. 39A) and SST, GHRL (FIG. 39B) that describe the effect of high exogenous glucose content during phase 6 in endocrine hormone expression. See Example 21.

FIG. 40A-B is a photographic micrograph showing the aggregates of endocrine cells of phase 7 as described in Example 21 with a high exogenous glucose content during phase 7. FIG. 40A shows staining for INS (cytoplasmic) and FIG. 40B for GCG (cytoplasmic).

FIG. 41 shows a flow cytometry graph showing the enrichment of immature phase 7 beta endocrine cells first treated with Py1, a zinc binding agent, and is classified by fluorescence. See Example 24.

FIG. 42A-C are bar graphs that show Nanostring mRNA data of the relative gene expression levels of INS, GCG, GHRL, IAPP, SST and SST (FIG. 42A); PAX4, PDX1, ARX, NKX6.1 (FIG. 42B); and PCSK1, GCK, G6PC2 and SLC30A8 (FIG. 42C) that characterize and identify the enriched cells from the zinc type. See Example 24.

FIG. 43 is a schematic diagram of phases 1-4 for the production of pancreatic endodermal cells (PEC) in vitro, and the development and maturation of insulin secreting beta cells in vivo. The diagram also shows the two main subpopulations of PEC, endocrine (CHGA +) and non-endocrine (CHGA-) cells. Embryonic stem cells (ESC), mesendoderm (ME), definitive endoderm (ED), upper digestive tract (FG), posterior superior digestive tract (pFG) and pancreatic epithelium or pancreatic endoderm (PE).

FIG. 44 is a schematic diagram of phases 1-7 for the production of endocrine cells in vitro. The diagram also shows that high levels of Activin (phase 3) followed by low levels of Activin and the elimination of WNT (phase 4) repress or inhibit the expression of NGN3, which is then expressed during phase 5 with the addition of a gamma secretase inhibitor. Embryonic stem cells (ESC), mesendoderm (ME), definitive endoderm (ED), upper digestive tract (FG), posterior superior digestive tract (pFG) and pancreatic epithelium or pancreatic endoderm (PE).

FIG. 45 is a schematic diagram of phases 1-7 for the production of endocrine cells in vitro. The diagram also shows the phases, growth factors, cell types and distinctive markers of each cell type: embryonic stem cells (ESC), mesendoderm (ME), definitive endoderm (ED), upper digestive tract (FG), Posterior upper digestive tract (pFG), pancreatic epithelium or pancreatic endoderm (PE), pre-beta cell (pre-B; or immature beta cell) and beta cell (p cell).

Detailed description of the invention

The present invention can be understood more quickly with reference to the following detailed description of the preferred embodiments of the invention and the examples included therein. However, before the present compounds, compositions and methods are disclosed and described, it should be understood that the present invention is not limited to specific cell types, specific feeder cell layers, specific conditions or specific methods, etc., and as such , may vary. Numerous modifications and variations in it will be apparent to those skilled in the art. It should also be understood that the terminology used herein is solely for the purpose of describing specific embodiments, and is not intended to be limiting.

Various cellular compositions derived from pluripotent stem cells are described herein, and can be found in US patent applications. UU. of applicant no .: 10 / 486.408, entitled "MEt Ho DS Fo R CULTURE OF HESC ON FEEDER CELLS", filed on Tuesday, August 6, 2002; 11,021,618, entitled "DEFINITIVE ENDODERM", presented on Thursday, December 23, 2004; 11 / 115,868, entitled "PDX1 EXPRESSING ENDODERM", presented on Tuesday, April 26, 2005; 11 / 165,305, entitled "METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATING DEFINITIVE ENDODERM", presented on Thursday, June 23, 2005; 11 / 573,662, entitled "METHODS FOR INCREASING DEFINITIVE ENDODERM DIFFERENTIATION OF PLURIPOTENT HUMAN EMBRYONIC STEM CELLS WITH PI-3 KINASE INHIBITORS", presented on Monday, August 15, 2005; 12 / 729,084, entitled "PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM", presented on Thursday, October 27, 2005; 12 / 093,590, entitled "MARKERS OF DEFINITIVE ENDODERM", presented on Monday, November 14, 2005; 11 / 993,399, entitled "EMBRYONIC STEM CELL CULTURE COMPOSITIONS AND METHODS OF USE THEREOF", presented on Tuesday, June 20, 2006; 11 / 588,693, entitled "PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM", presented on Friday, October 27, 2006; 11 / 681,687, entitled "ENDOCRINE PROGENITOR / PRECURSOR CELLS", "PANCREATIC HORMONE-EXPRESSING CELLS AND METHODS OF PRODUCTION", presented on Friday, March 2, 2007; 11 / 807,223, entitled "METHODS FOR CULTURE AND PRODUCTION OF SINGLE CELL POPULATIONS OF HESC", presented on Thursday, May 24, 2007; 11 / 773,944, entitled "METHODS OF PRODUCING PANCREATIC HORMONES", presented on Thursday, July 5, 2007; 11 / 860,494, entitled "METHODS FOR INCREASING DEFINITIVE ENDODERM PRODUCTION", presented on Monday, September 24, 2007; 12 / 099,759, entitled "METHODS OF PRODUCING PANCREATIC HORMONES", presented on Tuesday, April 8, 2008; 12 / 107.020, entitled "METHODS FOR PURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FORM HUMAN EMBRYONIC STEM CELLS", presented on Monday, April 21, 2008; 12 / 618,659, entitled "ENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS", presented on Friday, November 13, 2009; 12 / 765,714 and 13 / 761,078, both entitled "CELL COMPOSITIONS FROM DEDIFFERENTIATED REPROGRAMMED CELLS", filed on April 22, 2010 and February 6, 2013; 11 / 838,054, entitled "COMPOSITIONS AND METHODS USEFUL FOR CULTURING DIFFERENTIABLE CELLS", presented on Monday, August 13, 2007; 12 / 264,760, entitled "STEM CELL AGGREGATE SUSPENSION COMPOSITIONS AND METHODS OF DIFFERENTIATION THEREOF", presented on Tuesday, November 4, 2008; 13 / 259.15, entitled "SMALL MOLECULES SUPPORTING PLURIPOTENT CELL GROWTH", presented on Tuesday, April 27, 2010; PCT / US11 / 25628, entitled "LOADING SYSTEM FOR AN ENCAPSULATION DEVICE", presented on Monday, February 21, 2011; 13 / 992,931, entitled "AGENTS AND METHODS FOR INHIBITING PLURIPOTENT STEM CELLS", presented on Tuesday, December 28, 2010; and the US design requests No .: 29 / 408,366, filed on December 12, 2011; 29 / 408,368, filed on December 12, 2011; 29 / 423,365, filed on Thursday, May 31, 2012; No. 29 / 447,944, filed on Wednesday, March 13, 2013; and provisional US applications No .: 61 / 774,443, entitled "SEMIPERMEABLE MACRO IMPLANTABLE CELLULAR ENCAPSULATION DEVICES", presented on Thursday, March 7, 2013; 61 / 775,480, entitled "CRYOPRESERVATION, HIBERNATION AND ROOM TEMPERATURE STORAGE OF ENCAPSULATED PANCREATIC ENDODERM CELL AGGREGATES", presented on Friday, March 8, 2013; and 61 / 781.005, entitled "IN VITRO DIFFERENTIATION OF PLURIPOTENT STEM CELLS TO PANCREATIC ENDODERM CELLS (PEC) AND ENDOCRINE CELLS", presented on Thursday, March 14, 2013.

Definitions

It will be appreciated that the numerical ranges expressed herein include the established endpoints and describe all integers between the endpoints of the established numerical range.

Unless otherwise indicated, the terms used in this document should be understood in accordance with conventional use by experts in the relevant field. Also, for the purposes of this specification and the appended claims, unless otherwise indicated, it is understood that all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions and other numerical values used in The specification and in the claims should be understood as modified in all cases by the term "approximately". Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending on the desired properties that are sought to be obtained by the present invention. At a minimum, and not in an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter must be interpreted at least in the light of the number of significant digits indicated and by the application of usual rounding techniques.

The practice of the embodiments described herein employs, unless otherwise indicated, conventional techniques of cell biology, molecular biology, genetics, chemistry, microbiology, recombinant DNA and immunology.

The term "cell", as used herein, also refers to individual cells, cell lines or cultures derived from said cells. A "culture" refers to a composition comprising isolated cells of the same or a different type. "Cultivation", "population" or "cell population", as used herein, can be and are used interchangeably, and their meaning will be evident depending on the context. For example, the term "population" may be a cell culture of more than one cell having the same identification characteristics or it may be a culture of more than one type of cell having different identification characteristics, for example, a population, in one context, it can be a subpopulation in another context. The term "subpopulation" refers to a subset of a culture or a population of cells when used to describe certain types of cells within the cell culture or cell population.

As used herein, the term "totipotent stem cells" refers to cells that have the ability to differentiate into all cells that constitute an organism, such as cells that are produced from the fusion of an egg. and a sperm. The cells produced by the first divisions of the fertilized egg can also be totipotent. These cells can be differentiated into embryonic and extraembryonic cell types. Pluripotent stem cells, such as ES cells, for example, can give rise to any type of fetal or adult cells. However, they cannot become a fetal or adult animal alone, because they lack the potential to develop extraembryonic tissue. The extraembryonic tissue is, in part, derived from the extraembryonic endoderm, and can also be classified into parietal endoderm (Reichert's membrane) and visceral endoderm (part of the yolk sac). Both the parietal and visceral endoderm support embryo developments, but do not form embryonic structures. There are also other extra-embryonic tissues, which include the extra-embryonic mesoderm and the extra-embryonic ectoderm.

In some embodiments, a "pluripotent cell" is used as a starting material for differentiation to the endodermal lineage, or more particularly, to pancreatic endoderm type cells. As used herein, "pluripotence" or "pluripotent cells" or equivalent thereof refers to cells that are capable of proliferating in cell culture and differentiating into several populations of restricted lineage cells that have multipotent properties. For example, both pluripotent Es cells and induced pluripotent stem cells (iPS) can give rise to each of the three embryonic cell lineages. Pluripotent cells, however, are not able to produce a complete organism. That is, pluripotent cells are not totipotent.

In certain embodiments, the pluripotent cells used as starting material are stem cells, including hES cells, hEG cells, iPS cells, even parthenogenic cells and the like. As used herein, "embryonic" may refer to a range of phases of the development of an organism that begins with a single zygote and ends with a multicellular structure that no longer comprises pluripotent or totipotent cells other than the developed gamnetic cells. Pluripotent cells are not derived or not immediately derived from embryos, for example, iPS cells are derived from a non-pluripotent cell, for example, a multipotent cell or terminal differentiated cell. The human pluripotent stem cell lines used herein include hESC and iPSC, or any of those listed in Table 4.

Human pluripotent stem cells can also be defined or characterized by the presence of various transcription factors and cell surface proteins, including the Oct-4, Nanog and Sox-2 transcription factors, which form the central regulatory complex that guarantees the suppression of genes that lead to differentiation and maintenance of pluripotency; and cell surface antigens, such as glycolipids SSEA3, SSEA4 and keratin sulfate antigens, Tra-1-60 and Tra-1-81, and alkaline phosphatase.

As used herein, the term "induced pluripotent stem cells" or "iPS cells" or "iPSC" refers to a type of pluripotent stem cell artificially prepared from a non-pluripotent cell, usually a somatic cell adult or terminally differentiated cell, such as a fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell or the like, inserting certain genes or genetic products, called reprogramming factors. See Takahashi et al., Cell 131: 861-872 (2007); Wernig et al., Nature 448: 318-324 (2007); Park et al., Nature 451: 141-146 (2008). These and other subsequent known methods for the preparation of iPSC are well known, and the way in which iPSC is derived or produced is not limiting of the present invention. Induced pluripotent stem cells are essentially similar to natural human pluripotent stem cells, such as hES cells, in many aspects including, the expression of certain genes and stem cell proteins, chromatin methylation patterns, duplication time, formation of embryonic bodies, formation of teratomas, formation of viable chimeras, and power and capacity of differentiation. Human iPS cells provide a source of pluripotent stem cells without the associated use of embryos.

As used herein, the term "reprogramming", "reprogramming" or its equivalents, refers to a process that gives a cell considerably greater capacity to form a progeny of at least one new type of cell, either in culture or in vivo, of what it would have in the same conditions without reprogramming. In certain embodiments described herein, somatic cells are "reprogrammed" to pluripotent cells. In certain aspects, somatic cells are reprogrammed when, after sufficient proliferation, a measurable proportion of cells, either in vivo or in an in vitro cell culture , shows phenotypic characteristics of the new pluripotent cell type. Without reprogramming, these somatic cells would not give rise to a progeny that had phenotypic characteristics of the new type of pluripotent cell. If, even without reprogramming, somatic cells could lead to a progeny with phenotypic characteristics of the new type of pluripotent cell, the proportion of the progeny of these somatic cells showing phenotypic characteristics of the new type of pluripotent cell is considerably greater than before reprogramming

As used herein, the term "differentiation programming" refers to a process that changes a cell to form a progeny of at least one new cell type with a new state of differentiation, either in culture or in vivo. , which would have the same conditions without reprogramming the differentiation. This process includes differentiation, dedifferentiation and transdifferentiation. Therefore, as used herein, the term "differentiation" refers to the process by which a less specialized cell becomes a more specialized type of cell. On the contrary, the expression "dedifferentiation" refers to a cellular process in which a partially or terminal differentiated cell returns to an earlier stage of development, such as a cell that has pluripotence or multipotence. In greater contrast, the term "transdifferentiation" refers to a process of transforming a differentiated cell type into another differentiated cell type.

As used herein, "multipotence" or "multipotent cell" or its equivalents refers to a type of cell that can result in a limited number of other particular cell types. That is, multipotent cells engage with one or more embryonic cell destinations, and therefore, in contrast to pluripotent cells, cannot give rise to each of the three embryonic cell lineages, nor to extraembryonic cells. Multipotent somatic cells are more differentiated in relation to pluripotent cells, but do not differ terminally. Pluripotent cells, therefore, have greater potency than multipotent cells. Power determining factors that can reprogram somatic cells or that are used to generate iPS cells include, but are not limited to, factors such as Oct-4, Sox2, FoxD3, UTF1, Stella, Rex1, ZNF206, Sox15, Myb12, Lin28, Nanog, DPPA2, ESG1, Otx2 or combinations thereof.

As used herein, "unipotent" or "unipotent" or "unipotent cell" or its equivalents, refers to a type of cell that can give rise to a single cell of another lineage. For example, immature beta cells as described herein have the ability to differentiate only into insulin beta cells, and do not have the potential to differentiate into glucagon (alpha) cells, somatostatin (delta) cells and pancreatic polypeptide (gamma), for example. In contrast, endocrine precursor cells, PDX1-positive pancreatic endodermal cells, definitive endodermal cells or mesenddermal cells, and hES cells are all multipotent or pluripotent cells (hESC) that can give rise to each of the cells between pancreatic islets alpha, beta, delta and gamma. Similarly, the alpha, beta, delta and gamma pancreatic islet cells are endocrine precursor cell lineage cells, PDX1 positive pancreatic endodermal cells, definitive endodermal cells or mesedermal cells, and hES cells.

As used herein, "single hormone" and "polyhormonal" cells refer to cells that express only one hormone (eg, immature beta cells and beta cells express only insulin protein, and not glucagon or somatostatin protein), or express more than one or multiple hormones (for example, endocrine precursor cells or progenitor cells have subpopulations of cells that express 2, 3 or 4 or more hormones in the same cell). As used herein, the "ERBB receptor tyrosine kinase activating agent" includes, but is not limited to, at least 16 different EGF family ligands that bind to ERBB receptors: e Gf (factor of epidermal growth), AG or AREG (Anfirregulina) and TGF-Alfa (transforming growth factor alpha), Btc (Betacellulin), HBEGF (EGF binding to heparin) and Ereg (epirregulin), Neuregulins (or Heregulins) such as Neuregulin -1, -2, -3 and -4 (or Heregulin-1, -2, -3 and -4). However, the present invention contemplates any ligand that is capable of binding to any one of the four ERBB receptors or a combination thereof to induce the formation of homodimeric and heterodimer receptor complexes that lead to the activation of the kinase domain. intrinsic and subsequent phosphorylation. See also Table 13.

The term "cell lineage," as used herein, refers to all phases of the development of a cell type, from the oldest precursor cell to a fully mature cell (ie, a specialized cell). For example, a "definitive endodermal lineage cell" or a "PDX1 negative endodermal lineage cell" or a "PDX1 positive pancreatic endodermal lineage cell" or an "endocrine precursor lineage cell" or an "endocrine lineage cell "or an" immature beta lineage cell "and the like refer to cells derived from or differentiated from a definitive endodermal cell, a negative endodermal cell in PDX1, a pancreatic positive endodermal cell in PDX1 and the like. A definitive endodermal cell is a lineage of a mesedermal cell, one of its precursors. A positive pancreatic endodermal cell in PDX-1 is a lineage of a definitive endodermal cell, one of its precursors. An endocrine precursor in the lineage of a pancreatic cell positive in PDX1, a definitive endodermal cell and a mesededermal cell, all are its precursors. An immature beta cell in a lineage of an endocrine precursor cell, a PDX1 positive pancreatic cell, a definitive endodermal cell and a mesodermal cell, all are its precursors. A beta cell is the only lineage, for example, of an immature beta cell. However, all cells of the endodermal lineage described herein are cells of the hES lineage.

A "precursor cell" or "progenitor cell", as used herein, in general, can be any cell of a cell differentiation pathway that is capable of differentiating into a more mature cell. As such, a precursor cell can be a pluripotent cell, or it can be a partially differentiated multipotent cell or a reversibly differentiated cell. The term "population of precursor cells" refers to a group of cells capable of developing in a more mature or differentiated cell type. A population of precursor cells may comprise cells that are pluripotent, cells that have a restricted stem cell lineage (i.e., cells capable of developing in less than all ectodermal lineages, or in, for example, only neuronal lineage cells), and cells that are reversibly restricted to the stem cell lineage. Therefore, the term "progenitor cell" or "precursor cell" can be a "pluripotent cell" or "multipotent cell".

An "endocrine progenitor / precursor cell", as used herein, refers to a multipotent cell of the definitive endodermal lineage that expresses at least one marker on the list consisting of neurogenin 3 (NEUROG3), PDX1, PTF1A, SOX9 , NKX6.1, HNF1b, GATA4, HNF6, FOXA1, FOXA2, GATA6, MYT1, ISLET1, NEUROD, SNAIL2, MNX1, IA1, RFX6, PAX4, PAX6, NKX2.2, MAFA and MAFB, which can be further differentiated into cells of the endocrine system, including, but not limited to, cells that express the hormone of the pancreatic islet. Endocrine progenitor / precursor cells cannot be differentiated into so many different cells, tissue types and / or organs compared to definitively differentiated definitive endodermal lineage cells, such as PDX1-positive pancreatic endodermal cells or definitive endodermal cells or cells. mesedermal cells. Endocrine progenitor / precursor cells are described in detail in at least US Pat. UU. No. 8,129,182.

As used herein, the terms "develop from pluripotent cells", "differentiate from pluripotent cells", "mature from pluripotent cells" or "produced from pluripotent cells", "derive from pluripotent cells""," differentiate from pluripotent cells "and the equivalent expressions refer to the production of a differentiated cell type from pluripotent cells in vitro or in vivo, for example, in the case of mature endocrine cells from pancreatic endodermal cells PDX1 transplanted in vivo as described in International Patent Application No. PCT / US2007 / 015536, entitled "METHODS OF PRODUCING PANCREATIC HORMONES". All these terms and expressions refer to the progression of a cell from the phase of having the potential to differentiate into at least two different cell lines to become a specialized and terminal differentiated cell. These terms may be used interchangeably for the purposes of this application. The embodiments described herein contemplate culture conditions that allow such differentiation to be reversible, so that the pluripotency or at least the ability to differentiate into more than one cell lineage can be selectively recovered.

The term "feeder cell" refers to a cell culture that grows in vitro and secretes at least one factor in the culture medium, and that can be used to support the growth of another cell of interest in the culture. As used herein, a "feeder cell layer" can be used interchangeably with the term "feeder cell." A feeder cell can comprise a monolayer, in which the feeder cells cover the surface of the culture plate with a full layer before growing on top of each other, or they can comprise groups of cells. In a preferred embodiment, the feeder cell comprises an adherent monolayer.

Similarly, embodiments in which pluripotent cell cultures or aggregated pluripotent suspension cultures are grown under defined culture conditions or systems without the use of feeder cells are "free of feeder cells." Culture methods exempt from feeder cells increase the scalability and reproducibility of the pluripotent cell culture and reduce the risk of contamination, for example, by means of infectious agents of the feeder cells or other culture components of animal origin. Methods free of feeder cells are also described in US Pat. UU. No. 6,800,480 to Bodnar et al., (assigned to Geron Corporation, Menlo Park, California). However, and in contrast to US Pat. No. 6,800,480, the embodiments described herein, whether they are cultures of pluripotent, multipotent or differentiated cells, are exempt from feeder cells and do not further contain an endogenous or exogenous extracellular matrix; that is, the cultures described herein are free of extracellular matrix, in addition to being free of feeder cells. For example, in US Pat. No. 6,800,480, the extracellular matrix is prepared by culturing fibroblasts, lysing the fibroblasts in situ and then washing what is left after lysis. As an alternative, in US Pat. No. 6,800,480, the extracellular matrix can also be prepared from an isolated matrix component or a combination of components selected from collagen, placental matrix, fibronectin, laminin, merosine, tenascin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan, biglican, thrombospondin, vitronectin and decorin. The embodiments described herein neither produce an extracellular matrix by growing a feeder or fibroblast layer and lysing the cells to produce the extracellular matrix; nor do they first require coating the tissue culture vessel with an extracellular matrix component or a combination of extracellular matrix components selected from collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan , biglican, thrombospondin, vitronectin and decorin. Therefore, suspension cultures of aggregates described herein for pluripotent, multipotent and differentiated cells do not require a feeder layer, a lysed feeder or fibroblast cell to produce an extracellular matrix coating, an extracellular matrix or exogenously added matrix component. ; instead, the use of a soluble human serum component as described in the international application PCT / US2008 / 080516, entitled "METHODS AND COMPOSITIONS FOR FEEDER-FREE PLURIPOTENT STEM CELL MEDIA CONTAINING HUMAN SERUM", covers the need for a feeder cell or a feeder monolayer, while covering the need for an endogenous extracellular matrix of a feeder or fibroblast cell or exogenously added extracellular matrix components.

As used herein, the terms "grouping" and "group" or "aggregate" may be used interchangeably, and, in general, refer to a group of cells that have not dissociated into individual cells and which are then they have added forming clusters or, that have close contact from cell to cell. The term "reintegrate", as used herein, refers to when groupings, groups and / or aggregates they dissociate into smaller clusters, groups and / or aggregates, or individual cells and then form new cell-to-cell contacts by aggregation into clusters, groups and / or aggregates. This dissociation is usually of a manual nature (such as using a Pasteur pipette), but other means of dissociation are contemplated. The cultures of aggregated pluripotent or multipotent cells in suspension are essentially as described in the international publications PCT / US2007 / 062755, entitled "COMPOSITIONS AND METHODS FOR CULTURING DIFFERENTIAL CELLS" and PCT / US2008 / 082356, entitled "STEM CELL AGGREGATE SUSPENSION AND COMPOSITIONS COMPOSITIONS METHODS OF DIFFERENTIATION THEREOF ".

The term "single cell suspension" or its equivalents refers to a suspension of a single pluripotent, multipotent or terminal differentiated cell, or a suspension of a single cell derived from a pluripotent or multipotent cell, by any mechanical or chemical means. There are several methods to dissociate cell clusters to form suspensions of a single cell of primary tissues, cells bound in culture and aggregates, for example, physical forces (mechanical dissociation such as cell scraper, crushing through a narrow bore pipette, fine needle aspiration, vortex disaggregation and forced filtration through a fine nylon or stainless steel mesh), enzymes (enzymatic dissociation such as trypsin, collagenase, Accutase ™ and the like), or a combination of both. In addition, methods and conditions of culture media capable of supporting the dissociation into individual cells of pluripotent, multipotent or differentiated cells are useful for expansion, cell sorting and sowing defined for multi-well plate assays and allow the automation of cultivation procedures and clonic expansion.

In preferred embodiments, culture methods or culture systems are exempt from products of animal origin. In another preferred embodiment, the culture methods are free of xeno. In even more preferred embodiments, one or more conditions or requirements for the commercial manufacture of human cell therapeutic products met or exceeded the culture methods described herein.

The population of pluripotent cells can also be cultured in the presence of certain supplementary growth factors to obtain a population of cells that develop or develop in different cell lineages, or can be selectively reversed to be able to develop in different cell lineages. The term "supplementary growth factor" is used in its broadest context, and refers to a substance that is effective in enhancing the growth of a pluripotent cell, maintaining the survival of a cell, stimulating the differentiation of a cell and / or Stimulate the reversal of cell differentiation. In addition, a supplementary growth factor can be a substance that is secreted by a feeder cell in its environment. Such substances include, but are not limited to, cytokines, chemokines, small molecules, neutralizing antibodies and proteins. Growth factors may also include intercellular signaling polypeptides, which control the development and maintenance of cells, as well as the shape and function of tissues. In preferred embodiments, the supplemental growth factor is selected from the group comprising the stem cell factor (SCF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), interleukin-6 (IL-6) in combination with the receptor of soluble interleukin-6 (IL-6R), a fibroblast growth factor (FGF), a bone morphogenetic protein (BMP), tumor necrosis factor (TNF) and granulocyte macrophage colony stimulating factor (GM-CSF) .

In certain processes to produce the cells as described herein, growth factors are removed from the cell culture or from the cell population after their addition. For example, the growth factor, such as Activin A, Activin B, GDF-8 or GDF-11 can be added and removed in about one day, about two days, about three days, about four days, about five days, approximately six days, approximately seven days, approximately eight days, approximately nine days or approximately ten days after its addition. In some embodiments, the differentiation factors are not removed from the cell culture itself, but its omission of the cell culture medium is a means of elimination, for example, the change from a cell culture medium containing Activin to one that did not contain Activina

Because the efficiency of the differentiation process can be adjusted by modifying certain parameters, including, but not limited to, cell growth conditions, growth factor concentrations and the duration of the culture steps, the differentiation procedures described herein. document can give rise to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95% or more than approximately 95% conversion of pluripotent cells, which includes induced pluripotent cells, to multipotent or differentiated cells, for example, definitive endoderm. On the other hand, the conversion rate or the efficiency rate can also refer to the differentiation of a multipotent cell type into an additional differentiated multipotent cell, for example, from definitive to endodermal endodermic of the upper digestive tract, endodermal of the upper digestive tract positive in PDX1, positive pancreatic endodermal in PDX1 or positive pancreatic endodermal in both PDX1 and NKX6.1, endocrine progenitor / precursor or endocrine progenitor / precursor positive in both NGN3 and NKX2.2 and endocrine hormone secreting cells or positive endocrine cells only in INS, GCG, GHRL, SST, PP. In the processes in which the isolation of mesenddermal cells or in preprimitive stretch marks is used, a population of mesenddermal cells or in pure preprimitive stretch marks can be recovered.

General methods for the production of endodermal lineage cells derived from hES cells are described in US applications. related as indicated above, and D'Amour et al. 2005 Nat Biotechnol.

23: 1534-41, published online Friday, October 28, 2005; D'Amour et al. 2006 Nat Biotechnol. 24 (11): 1392-401, published online Thursday, October 19, 2006; Kroon et al. (2008) Nat Biotechnol. 26 (4): 443-452, published online on Wednesday, February 20, 2008; Kelly et al. (2011) Nat. Biotechnol. 29 (8): 750-6, published online Sunday, July 31, 2011; and Schulz et al. (2012) PLosOne, 7 (5): e37004, published online on Friday, May 18, 2012. D'Amour et al. They describe a 5-stage differentiation protocol: phase 1 (gives rise to the majority of the final endodermal production), phase 2 (results in the majority of the endodermal production of the upper negative digestive tract in PDX1), phase 3 (results to most of the endodermal production of the upper positive digestive tract in PDX-1), phase 4 (gives rise to the majority of pancreatic or endocrine endocrine progenitor endodermal production) and phase 5 (gives rise to the majority of cell production endocrines that express hormones).

The term "troofectoderm" refers to a multipotent cell that has the high relative expression of markers selected from the group comprising genes HAND1, Eomes, MASH2, ESXL1, HCG, KRT18, PSG3, SFXN5, DLX3, PSX1, ETS2 and ERBB in comparison with the expression levels of HAND1, Eomes, MASH2, ESXL1, HCG, KRT18, PSG3, SFXN5, DLX3, PSX1, ETS2 and ERBB in cells or populations of non-troofectodermal cells.

The term "extra-embryonic endodermal" refers to a multipotent cell that has high relative expression levels of markers selected from the group comprising SOX7, SOX17, THBD, SPARC, DAB1 or AFP genes compared to SOX7, SOX17 expression levels, THBD, SPARC, DAB1 or AFP in cells or populations of non-extraembryonic endodermal cells.

The expression "cells in preprimitive stretch marks" refers to a multipotent cell that has high relative expression levels of the FGF8 and / or NODAL marker genes, compared to a low level of BRAQUIURIA, low level of FGF4, low level of SNAI1 , low level of SOX17, low level of FOXA2, low level of SOX7 and low level of SOX1.

The term "mesenddermal cell" refers to a multipotent cell that has high relative expression levels of brachyuria marker genes, FGF4, SNAI1 MIXL1 and / or WNT3, compared with low level of SOX17, low level of CXCR4, low level of FOXA2, low level of SOX7 and low level of SOX1.

The term "definitive endodermal cell (ED)" refers to a multipotent endodermal lineage cell that can be differentiated into intestinal tube cells or organs derived from the intestinal tube. According to certain embodiments, the definitive endodermal cells are mammalian cells, and in a preferred embodiment, the final endodermal cells are human cells. In some embodiments of the present invention, the final endodermal cells express or not express certain markers significantly. In some embodiments, one or more selected markers of SOX17, CXCR4, MIXL1, GATA4, HNF3p, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 are expressed in definitive endodermal cells. In other embodiments, one or more markers selected from OCT4, alpha-fetoprotein (AFP), Thrombomodulin (TM), SPARC, SOX7 and HNF4alpha are not expressed or expressed significantly in definitive endodermal cells. The final endodermal cell populations and their production methods are also described in the US application. UU. No. 11 / 021,618, entitled "DEFINITIVE En Do DERM", presented on Thursday, December 23, 2004.

The expression "endodermal cells of the upper digestive tract negative in PDX1" or "endodermal cells of the upper digestive tract" or equivalent thereof are cells that express SOX17, HNF1p (HNF1B), HNF4alfa (HNF4A) and FOXA1 markers, but do not substantially express PDX1, AFP, SOX7 or SOX1. The populations of endodermal cells of the upper digestive tract negative in PDX1 and their production methods are also described in US application No. 11 / 588,693, entitled "PDX1-expressing dorsal and ventral foregut endoderm" , filed on Friday, October 27, 2006.

The expression "endodermal cells of the upper digestive tract, with dorsal bias, positive in PDX1" (endodermal cells of the upper digestive tract positive in dorsal PDX1) or simply "endodermal positive in PDX1" or the equivalents thereof are cells expressing one or more more markers selected from Table 1 and / or one or more markers selected from Table 2, also described in the US application. UU. related 11 / 588,693, entitled "PDX1 EXPRESSING DOSAL AND VENTRAL FOREGUT ENDODERM", filed on October 27, 2006 and also in the US application. No. 11 / 115,868, entitled "PDX1-expressing endoderm", filed on Tuesday, April 26, 2005.

Table 1: Markers expressed in positive upper digestive tract endoderm in both dorsal and ventral PDX1

(continuation)

T l 2: M r r x r n n rm i iv ri r iiv n PDX1 n r l.

(continuation)

The expression "pancreatic endoderm""pancreaticepithelium""pancreaticepithelium" (all may be abbreviated as "PE"), "pancreatic progenitor", "positive pancreatic endoderm in p DX-1" or its equivalents, such as "pancreatic endodermal cells" (PEC), are all precursor or progenitor pancreatic cells. The PEC as described herein is a population of progenitor cells subsequent to phase 4 differentiation (approximately day 12-14), and includes at least two distinct major populations: i) pancreatic progenitor cells expressing PDX1 and NKX6. 1, but do not express CHGA (or negative in CHGA, CHGA-), or "multipotent non-endocrine progenitor subpopulations (CHGA-)", or "non-endocrine subpopulations (CHGA-)" or "non-endocrine cells (CHGA-)" or equivalents thereof; and ii) polyhormonal endocrine cells expressing CHGA (positive in CHGA, CHGA +) or "multipotent endocrine progenitor subpopulations (CHGA +)", or "endocrine subpopulations (CHGA +)" or "endocrine cells (CHGA +)" or equivalent thereof. The PEC pancreatic progenitor subpopulation that expresses PDX1 and NKX6.1, but does not express CHGA is also known as "non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-)" or "non-endocrine progenitor subpopulation", "non-endocrine subpopulation (CHGA-)" , "non-endocrine subpopulation (CHGA-)", "multipotent progenitor subpopulation" and the like. The subpopulation of PEC polyhormonal endocrine cells that expresses CHGA is also known as "cells committed to the endocrine lineage (CHGA +)" or "endocrine cells" or "CHGA + cells" and the like. Without being bound to any theory, the cell population that expresses NKX6.1, but not CHGA, is supposed to be the most active or therapeutic component of PEC, while the population of positive polyhormonal endocrine cells in CHGA is assumed to be more different and matures in vivo in islet cells that express glucagon.

See Kelly et al. (2011) "Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells", Nat Biotechnol. 29 (8): 750-756, published online July 31, 2011 and Schulz et al. (2012), "A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells", PLosOne 7 (5): 1-17, e37004.

Even so, sometimes, pancreatic endodermal cells are used without reference to PEC as just described, but to refer to at least phase 3 and 4 cells in general. The use and meaning will be evident from the context. The pancreatic endoderm derived from pluripotent stem cells, and at least hES and hIPS cells, in this way, is distinguished from other types of endodermal lineage cells depending on the differential or high expression levels of markers selected from PDX1, NKX6 marker. 1, PTF1A, CPA1, cMYC, NGN3, PAX4, ARX and NKX2.2, but do not substantially express genes that are the hallmark of pancreatic endocrine cells, for example, CHGA, INS, GCG, GHRL, SST, MAfA, PCSK1 and GLUT1. In addition, some "endocrine progenitor cells" that express NGN3 may differentiate into other non-pancreatic structures (eg, duodenum). Populations of endocrine pancreatic or endocrine progenitor cells, and their methods are also described in US Pat. No. 11 / 773,944, entitled "Methods of producing pancreatic hormones," filed July 5, 2007 and U.S. Patent Application. No. 12 / 107.020, entitled "METHODS FOR PURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FORM HUMAN EMBRYONIC STEM CELLS", presented on Monday, April 21, 2008.

The expression "endocrine cell" or "cell that expresses the hormone of the pancreatic islet", "pancreatic endocrine cell", "cell of the pancreatic islets", "pancreatic islets" or equivalent thereof refers to a cell, which may be polyhormonal or single hormone. The cells can, therefore, express one or more pancreatic hormones that have at least some of the functions of a human pancreatic islet cell. Cells expressing pancreatic islet hormones may be mature or immature, and subsequently differentiated or subsequently evolved in comparison to an endocrine progenitor / precursor cell from which they are derived.

As used herein, the term "suitably specified endocrine cells" or "phase 7 cultures" or "immature endocrine cells" that include "immature beta cells" refers to populations of in vitro manufactured endocrine cells that are capable of function in vivo, for example, immature beta cells, when transplanted, secrete insulin in response to blood glucose. Endocrine cells or properly specified phase 7 cultures may have additional characteristics that include the following: when transplanted, properly specified endocrine cells can develop and mature to become functional cells of the pancreatic islets. Properly specified endocrine cells can be enriched in endocrine cells (or depleted of non-endocrine cells). In a preferred embodiment, more than 50% of the cells of the properly specified endocrine cell population are CHGA +. In a more preferred embodiment, more than about 60% or 70% or 80% or 90% or 95% or 98% or 100% of the cells of the appropriately specified endocrine cell population are CHGA +. In a preferred embodiment, less than about 50% of the cells of the properly specified endocrine cell population are CHGA-. In a more preferred embodiment, less than about 15% of the cells of the properly specified endocrine cell population are CHGA-. In a more preferred embodiment, less than about 10% or 5% or 3% or 2% or 1% or 0.5% or 0% of the cells of the properly specified endocrine cell population are CHGA-. In addition, the expression of certain markers can be suppressed in duly specified endocrine cells, such as the expression of NGN3 during phase 3. Suitably specified endocrine cells may have a greater expression of NGN3 in phase 5. Properly specified endocrine cells can be of a single hormone (for example, only INS, only GCG or only SST). Properly specified endocrine cells can jointly express other immature endocrine cell markers, including NKX6.1 and PDX1. Properly specified endocrine cells can both express a single hormone and jointly express other immature endocrine cell markers, including NKX6.1 and PDX1. Properly specified endocrine cells may have more cells that only express the INS hormone as a percentage of the total INS population. In a preferred embodiment, properly specified endocrine cells have at least 50% of cells expressing a single INS hormone as a percentage of the total INS population. The properly specified endocrine cells can be CHGA + / INS + / NKX6.1 + (triple positive). In a preferred embodiment, more than about 25% of the cells of the cell population are CHGA + / INS + / NKX6.1 + (triple positive). In a preferred embodiment, more than about 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95% 100% of the cells of the cell population are CHGA + / INS + / NKX6.1 + ( triple positive).

The term "immature endocrine cell", specifically an "immature beta cell" or its equivalents refers to a cell derived from any other endocrine cell precursor that includes an endocrine progenitor / precursor cell, a pancreatic endodermal cell (PE), a cell pancreatic of the upper digestive tract, a definitive endodermal cell, a mesendodermal cell or any other derived cell described below, which expresses at least one marker selected from the group consisting of INS, NKX6.1, PDX1, NEUROD, MNX1, n Kx 2.2 , MAFA, PAX4, SNAIL2, FOXA1 or FOXA2. Preferably, an immature beta cell described herein expresses INS, NKX6.1 and PDX1, and more preferably jointly expresses INS and NKX6.1. The terms "immature endocrine cell", "immature cell expressing pancreatic hormones" or "pancreatic islet immature "or its equivalents refer, for example, to at least one immature unipotent beta cell or pre beta cell as described in FIG. 45, and do not include other immature cells, for example, the expressions do not include an alpha cell ( glucagon) immature or an immature delta cell (somatostatin) or an immature epsilon cell (ghrelin) or an immature pancreatic polypeptide (PP).

In the embodiments described herein, many culture media or stem cell growth environments are contemplated, including defined media, conditioned media, feeder cell-free media, serum-free media and the like. As used herein, the term "growth environment" or "environment" or equivalent thereof is an environment in which undifferentiated or differentiated stem cells (eg, embryonic primate stem cells) will proliferate in vitro. . The characteristics of the environment include the medium in which the cells are grown and a support structure (such as a substrate on a solid surface) if present. Methods for culturing or maintaining pluripotent cells and / or differentiating pluripotent cells are also described in document PCT / US2007 / 062755 entitled "COMPOSITIONS AND METHODS USEFUL FOR CULTURING DIFFERENTIABLE CELLS", filed on Friday, February 23, 2007; the US request No. 11 / 993,399, entitled "EMBRYONIC STEM CELL CULTURE COMPOSITIONS AND METHODS OF USE THEREOF", presented on Thursday, December 20, 2007; the US request No. 11/875,057, entitled "Methods and compositions for feeder-free pluripotent stem cell media containing human serum", filed on Friday, October 19, 2007.

The term "essentially" or "substantially" means, for the most part or a minimum or a reduced amount of a component or cell present in any cell population or culture, for example, the immature beta cell cultures described herein. they are "essentially or substantially cells" or are "essentially or substantially immature beta cells that express INS, NKX6.1 and PDX1, and that do not express essentially or substantially NGN3". Other examples include, but are not limited to, "Essentially or substantially HES cells," "Essentially or substantially defined endodermal cells," "Essentially or substantially superior endogenous digestive tract cells," "Essentially or substantially negative upper digestive tract endodermal cells in PDX1. "," Essentially or substantially positive pancreatic endodermal cells in PDX1, "" Essentially or substantially pancreatic endocrine precursor cells, "" Essentially or substantially pancreatic endocrine cells "and the like.

With respect to cell culture cells or cell populations, the term "substantially free of" means that the specified cell type from which the cell culture or cell population is exempt, is present in an amount less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less at about 2% or less than about 1% of the total number of cells present in the cell culture or cell population.

The term "reduced serum" or "serum free" or its equivalents refers to cell cultures grown in a medium that contains reduced serum or that is substantially serum free or that does not contain serum at all. Under certain culture conditions, serum concentrations may vary between about 0% (v / v) and about 10% (v / v). For example, in some differentiation processes, the serum concentration of the medium may be less than about 0.05% (v / v), less than about 0.1% (v / v), less than about 0, 2% (v / v), less than about 0.3% (v / v), less than about 0.4% (v / v), less than about 0.5% (v / v), less than about 0.6% (v / v), less than about 0.7% (v / v), less than about 0.8% (v / v), less than about 0.9% (v / v), less than about 1% (v / v), less than about 2% (v / v), less than about 3% (v / v), less than about 4% (v / v), less than about 5% (v / v), less than about 6% (v / v), less than about 7% (v / v), less than about 8% (v / v ), less than about 9% (v / v) or less than about 10% (v / v). In some processes, cells in preprimitive stretch marks are grown without serum or without serum replacement. In other processes, cells in preprimitive stretch marks are cultured in the presence of B27. In such processes, the concentration of supplement B27 may vary from about 0.1% (v / v) to about 20% (v / v).

In other processes, cell cultures are grown in the presence of B27. In such processes, the concentration of supplement B27 may vary from about 0.1% (v / v) to about 20% (v / v) or in concentrations greater than about 20% (v / v). In certain processes, the concentration of B27 in the medium is approximately 0.1% (v / v), approximately 0.2% (v / v), approximately 0.3% (v / v), approximately 0.4% (v / v), approximately 0.5% (v / v), approximately 0.6% (v / v), approximately 0.7% (v / v), approximately 0 , 8% (v / v), approximately 0.9% (v / v), approximately 1% (v / v), approximately 2% (v / v), approximately 3% (v / v) , approximately 4% (v / v), approximately 5% (v / v), approximately 6% (v / v), approximately 7% (v / v), approximately 8% (v / v) , approximately 9% (v / v), approximately 10% (v / v), approximately 15% (v / v) or approximately 20% (v / v). Alternatively, the concentration of added b27 supplement can be measured in terms of multiples of the concentration of a commercially available B27 stock solution. For example, B27 is available in Invitrogen (Carlsbad, CA) as an x50 stock solution. The addition of a sufficient amount of this stock solution to a sufficient volume of growth medium produces a medium supplemented with the desired amount of B27. For example, the addition of 10 ml of stock solution x50 of B27 to 90 ml of growth medium would produce a growth medium supplemented with B27 x5. The concentration of supplement B27 in the medium may be about x0.1, about x0.2, about x0.3, about x0.4, about x0.5, about x0.6, about x0.7, about x0.8 , approximately x0.9, approximately x1, approximately x1.1, approximately x1.2, approximately x1.3, approximately x1.4, approximately x1.5, approximately x1.6, approximately x1.7, approximately x1.8, approximately x1.9, about x2, about x2.5, about x3, about x3.5, about x4, about x4.5, about x5, about x6, about x7, about x8, about x9, about x10, about x11, about x12, approximately x13, approximately x14, approximately x15, approximately x16, approximately x17, approximately x18, approximately x19, approximately x20 and greater than approximately x20.

In another aspect, in which the insulin level requirements for differentiation are low, B27 is not used, because B27 contains high levels of insulin. For example, the applicant determined insulin levels in stock solutions of B27 and ITS x100 and in working stock solutions x1 of each in RPMI. Insulin levels were determined using a Mercodia ultrasensitive insulin ELISA kit according to the manufacturer's instructions. The tests were performed in x27 and x50 stock solutions of B27 and unopened STIs, respectively, both purchased from Life Technologies (Carlsbad, California). The results generated from the test are shown in Table 3 below.

Table 3 shows that there are approximately 3 jg / mly and 10 | jg / ml of insulin present in lxB27 and lxITS, respectively. In addition, the concentration of insulin in the ITS x100 stock solution coincides with the insulin concentration indicated by the manufacturer. Life Technologies does not provide the insulin concentration for B27 x50, so there is no comparison with the insulin levels indicated by the manufacturer; however, the previous insulin concentration of approximately 160 jg / ml is accurate based on the exact measurement of the STI x100 stock solution that was performed at the same time and according to McLean et al. (2007) supra, which showed that both insulin and insulin-like growth factor (IGF) are well established agonists of PI3-kinase-dependent signaling, suggesting that a PI3-kinase inhibitor (by For example, LY 294002) would enhance the definitive formation of the endoderm by inhibiting the effectors of insulin / IGF. See FIG.6A and B, specifically, the expression of SOX17 is reduced 4 times with 1 jg / ml insulin. Insulin and / or IGF are present at biologically active levels in various media supplements such as KSR, FCS (fetal calf serum), FBS (fetal bovine serum), B27 and StemPro34, and can inhibit definitive endoderm differentiation in such culture conditions, for example, Jiang et al. (2007) supra.

As used herein, "exogenous" or "exogenously added", compounds such as agents, components, growth factors, differentiation factors, and the like, in the context of crops or conditioned media, refers to what It is added to the cultures or media to complement any compound or growth factor that may or may not already be present in the culture or in the media. For example, in some embodiments, cell cultures and / or cell populations do not include an exogenously added retinoid.

As used herein, "retinoid" refers to retinol, retinal or retinoic acid, as well as derivatives of any of these compounds. In a preferred embodiment, the retinoid is retinoic acid.

The term "growth factor of the FGF family" "a fibroblast growth factor" or "member of the fibroblast growth factor family" means an FGF selected from the group comprising FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22 and FGF23. In some embodiments, "growth factor of the FGF family", "a fibroblast growth factor" or "member of the fibroblast growth factor family" means any growth factor that has similar homology and / or function to a known member of the family of fibroblast growth factors.

The term "TGFp superfamily member" or "TGFp superfamily ligand" or "TGFp TGF-beta signaling pathway activator" or equivalent thereof refers to more than 30 structurally related proteins, including subfamilies of TGF-beta1, TGF-beta2, TF-beta3, GDF-15, GDF-9, BMP-15, BMP-16, BMP-3, GDF-10, BMP-9, BMP-10, GDF-6, GDF-5, GDF-7, BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, BMP-4, GDF-3, GDF-1, GDF 11, GDF8, BetaC, betaE, betaA and betaB, BMP-14, GDF-14, MIS, Inhibin alfa, Leftyl, Lefty2, GDNF, Neurteurin, Persephine and Artemine. See Chang et al. (2002) Endocrine Rev. 23 (6): 787-823. These ligands are normally synthesized as prepropeptides of approximately 400-500 amino acids (aa), and a "TGFp superfamily member" or "TGFp superfamily ligand" or "TGFp TGF-beta signaling pathway activator" or its equivalents can be the full length protein or a proteolytic peptide thereof.

The term "ERBB ligand" refers to a ligand that binds to any of the ErbB receptors, which, in turn, can be dimerized with another ErbB receptor, or it can function as a monomer, thus activating the tyrosine kinase activity of the monomer of the ErbB portion or a dimer or a heterodimeric receptor. Non-limiting examples of ErbB ligands include neuregulin-1; splice variants and neurregulin-1 sophormas, including, but not limited to, HRG-p, HRG-a, Neu differentiation factor (NDF), acetylcholine receptor inducing activity (ARIA), growth factor glial 2 (GGF2) and factor derived from sensory and motor neurons (SMDF); Neurregulin-2; splice variants and isoforms of Neuregulin-2, including, but not limited to, NRG2-p antagonists; Epirregulin; and Birregulina.

The term "expression", as used herein, refers to the production of a material or a substance, as well as to the level or amount of production of a material or a substance. Therefore, the determination of the expression of a specific marker refers to detecting the relative or absolute amount of the marker that is expressed or simply detecting the presence or absence of the marker.

The term "marker", as used herein, refers to any molecule that can be observed or detected. For example, a label may include, but is not limited to, a nucleic acid, such as a transcription of a specific gene, a polypeptide product of a gene, a non-gene product polypeptide, a glycoprotein, a carbohydrate, a glycolipid, a lipid, a lipoprotein or a small molecule (for example, molecules that have a molecular weight less than 10,000 amu).

For most of the markers described herein, the official symbol of the Human Genome Organization (HUGO) gene is provided. These symbols, which are developed by the HUGO Nomenclature Committee, provide unique abbreviations for each of the named human gene products and genes. Those skilled in the art readily recognize these gene symbols and can easily be associated with a corresponding unique human gene and / or protein sequence.

According to the HUGO designations, the following gene symbols are defined as follows: GHRL-ghrelin; lAPP-amyloid islet polypeptide; INS - insulin; GCG-glucagon; ISL1 - ISL1 transcription factor; PAX6 - paired box gene 6; PAX4 - paired box gene 4; NEUROG3 - neurogenin 3 (NGN3); NKX2-2 - related NKX2 transcription factor, locus 2 (n Kx 2.2); NKX6-1 - related NKX6 transcription factor, locus 1 (NKX6.1); IPF1 - insulin promoting factor 1 (PDXl); ONECUT1 - a cutting domain, family member 1 (HNF6); HLXB9 - homeotic box B9 (HB9); TCF2 - transcription factor 2, hepatic (HNF1b); FOXA1- Forkhead A1 box; HGF - hepatocyte growth factor; IGF1 - insulin-like growth factor 1; POU5F1 - POU domain, class 5, transcription factor 1 (OCT4); NANOG - Nanog homeotic box; SOX2 - SRY (Y region for sex determination) - box 2; CDH1 - cadherin 1, type 1, E-cadherin (ECAD); T - Brachyuria counterpart (BRACH); FGF4 - fibroblast growth factor 4; WNT3 - family of MMTV integration sites of wingless type, member 3; SOX17 - SRY (sex determination region Y) - box 17; GSC - goosecoid; CER1 - (cerberus 1, cysteine knot superfamily, homolog (CER); CXCR4 - chemokine receptor 4 (CXC motif); FGF17 - fibroblast growth factor 17; FOXA2 - forkhead A2 box; SOX7 - SRY (Y region of sex determination) -box 7; SOX1-SRY (Y region for sex determination) -box 1; AFP-alpha-fetoprotein; SPARC - secreted, acidic, cysteine-rich protein (osteonectin); and THBD-thrombomodulin (TM) , NCAM - neuronal cell adhesion molecule; SYP - synaptophysin; ZIC1 - member 1 of the Zic family; NEF3 - neurofilament 3 (NFM); SST -somatostatin; MAFA - homologue A of the musculoaponeurotic fibrosarcoma oncogene v-maf; MAFB - V-maf musculoaponeurotic fibrosarcoma oncogene homologue B; SYP - synaptophysin; CHGA - chromogranin A (parathyroid secreting protein 1); NGN3 - Neurogenin 3, NKX2.2 - Homeotic box 2 of NK2; NKX6.1- Homeotic box 1 of NK6; ID1 - DNA binding inhibitor 1, GHRL - Ghrelin Prepropeptide / Obestatin, GSK - glycogen synthase kinase, G6PC2 - glucose-6-phosphatase, UCN3 - urocortin 3, PCSK1 - proprotein convertase subtilisin / kexin type 1, SLC30A8 - solute transporter family 30 (zinc carrier), member 8 and CADH1 - E-Cadherina.

The terms fibroblast growth factor 7 (FGF7) and keratinocyte growth factor (KGF) are synonyms.

The progression of pluripotent cells to several multipotent and / or differentiated cells can be monitored by determining the relative expression of genes or gene markers, characteristic of a specific cell, compared to the expression of a second gene or control gene, by example, constitutive genes. In some processes, the expression of certain markers is determined by detecting the presence or absence of the marker. Alternatively, the expression of certain markers can be determined by measuring the level at which the marker is present in the cells of the cell culture or the cell population. In such processes, the measure of marker expression can be qualitative or quantitative. One method to quantify the expression of markers produced by marker genes is through the use of quantitative PCR (Q-PCR). The methods for performing the Q-PCR are well known in the art. Other methods known in the art can also be used to quantify expression of the marker gene. For example, the expression of a marker gene product can be detected using antibodies specific to the marker gene product of interest.

In addition, multiplexing assays and / or methodologies are available to analyze many genes with high sensitivity and efficiency. For example, with at least the nCounter system provided by Nanostring (Seattle, WA, USA), users can now analyze the expression levels of up to 800 genes simultaneously, with a sensitivity comparable to that of the systems of quantitative PCR and with less than 15 minutes of practical time per reaction. Accordingly, the total RNA in any of the samples (for example, cultures of PEC cells in phase 4 and endocrine and endocrine progenitors / precursors in phase 7, respectively) can be isolated (for example, in triplicate) using the acid extractor nucleic 6100 (Applied Biosystems; Foster City, CA, USA); and approximately 100 ng per reaction is required for the quantification of gene expression using the nCounter Nanostring System. And instead of analog detection, the nCounter system uses digital detection, so each gene transcript is detected by a probe attached to a segment of DNA that is attached to a single chain of colored fluorophores (the barcode molecular). The identification of that transcript, therefore, depends only on the order of the fluorine in the chain, rather than the intensities of the fluorine. Second, the number of total transcripts in a sample is quantified by counting the total number of times a particular fluorine chain (barcode) is detected. In addition, this method does not require amplification of the target mRNA, therefore, the range is the biological range of expression, usually three orders of magnitude. Currently, the system can measure only 1.2 times the changes of a single transcript at 20 copies per cell (10 fM) with statistical significance ( p <0.05). For genes expressed at levels between 0.5 and 20 copies per cell, 1.5-fold differences in expression levels are detectable with the same level of confidence.

In some processes, the highest expression of the following genes compared to the lowest expression of other genes is indicative of certain cell populations, for example: SOX17, SOX7, AFP or THBD are indicative of extraembryonic endoderm; NODAL and / or FGF8 are indicative of preprimitive stretch marks; brachyuria, FGF4, SNAI1 and / or WNT3 are indicative of mesendoderm; CER, GSC, CXCR4, SOX17 and FOXA2 are indicative of definitive endodermal cells; SOX17, FOXA2, FOXA1, HNF1B and HNF4A are indicative of posterior digestive tract endoderm (or negative endoderm in PDX1); PDX1, HNF6, SOX9 and PROX1 are indicative of positive endoderm in PDX1; PDX1, NKX6.1, PTFA1, CPA and cMYC are indicative of pancreatic epithelium (PE or pancreatic parent); NGN3, PAX4, ARX and NKX2.2 are indicative of endocrine progenitor / precursor cells; and INS, Gc G, GHr L, SST and PP are indicative of the various endocrine cells; high relative expression of MAFA to MAFB genes is indicative of insulin secreting endocrine cells; and the high relative expression of MAFB with respect to the expression of the MAFA gene is indicative of glucagon-secreting endocrine cells. However, only "distinctive" or "key" markers typical of that particular cell type of a particular lineage are shown in certain figures and drawings. As the person skilled in the art will understand, those other markers or genes are expressed, but are not shown or described, for example, genes that are constitutively expressed or expressed in all or most or most of the cell types. of a certain lineage or all lineages.

Methods for the production of induced pluripotent stem cells (iPS)

The embodiments described herein are not limited to an iPS cell type or a method of producing the iPS cell. The embodiments are not limited or depend on the levels of production efficiency of the iPS cells, because there are several methods. The embodiments described herein apply to the differentiation of iPS cells into endodermal lineage cells and uses thereof.

Studies using certain nuclear reprogramming factors have allowed pluripotent stem cells or pluripotent-like stem cells to be derived from the patient's own somatic cells. These cells are also called pluripotent induced stem cells (iPS). The present invention describes various strains of iPS cells provided by Shinya Yamanaka, Kyoto University. However, other iPS cell lines, for example, those described by James Thomson et al., Are encompassed by the invention herein. See U.S. publication U. 20090047263, international publication WO2005 / 80598, U.S. publication

20080233610 and the international publication WO2008 / 11882. Therefore, as used herein, "induced pluripotent stem cells (iPS)" means cells that have similar properties to other pluripotent stem cells, for example, hES cells, hEG cells, pPS (pluripotent primate stem cells) cells parthenogenic and similar.

Nuclear programming factors are described in the US publication. 20090047263, the international publication WO2005 / 80598, the US publication 20080233610 and the international publication WO2008 / 11882, and were used to induce reprogramming of a differentiated cell without using ovules, embryos or ES cells. The methods for preparing iPS cells induced from somatic cells using the nuclear reprogramming factor similar to that used and described in the present invention are not particularly limited. In preferred embodiments, the nuclear reprogramming factor comes into contact with somatic cells in an environment in which somatic cells and induced pluripotent stem cells can proliferate. An advantage of the certain embodiments described herein is that an induced pluripotent stem cell can be prepared by contacting a nuclear reprogramming factor with a somatic cell in the absence of ovules, embryos or embryonic stem cells (ES). By using a nuclear reprogramming factor, the nucleus of a somatic cell can be reprogrammed, obtaining an iPS cell or an "ES-like cell".

Pluripotent stem cells described herein, whether hES cells or iPS cells, can express any number of pluripotent cell markers, including, but not limited to: alkaline phosphatase (AP); ABCG2; Embryonic phase-specific antigen 1 (SSEA-1); Ss Ea -3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin; .p.III-tubulin; smooth muscle a-actin (a-SMA); fibroblast growth factor 4 (Fgf4), Crypto, Dax1; Zinc finger protein 296 (Zfp296); N-acetyltransferase 1 (Nati); (transcription associated with ES 1 cells (ECAT1); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14; undifferentiated embryonic cell transcription factor (Utf1 ); Rex1; p53; G3PDH; telomerase, including TERT; silent X chromosome genes; Dnmt3a; Dnmt3b; TRIM28; Box F containing protein 15 (Fbx15); Nanog / ECAT4; Oct3 / 4; Sox2; Klf4; c-Myc ; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; associated developmental pluripotency 2 (DPPA2); T 1 lymphocyte rupture point (Tel1); DPPA3 / Stella; DPPA4 and the like. The invention is not limited to the markers listed herein, and encompasses markers such as cell surface markers, antigens and other gene products, including ESTs, RNA (including microRNA and antisense RNA), DNA (including genes and cDNA) and his parts.

In one embodiment, the iPS cell lines used herein contain the following nuclear reprogramming factor genes: a gene from the Oct family, a gene from the Klf family and a gene from the Sox family. In an iPS cell line, each of the following three types of genes are provided: Oct3 / 4, Klf4 and Sox2. Other iPS cell lineage products of each of the following three types of genes were used: a gene from the Oct family, a gene from the Klf family and a gene from the Myc family, for example, Oct3 / 4, Klf4 and c-Myc. Therefore, it is understood that the nuclear reprogramming factor can be with or without the Myc family gene.

The nuclear reprogramming factors described herein and also known in the art can be used to generate iPS cells from differentiated adult somatic cells, and are not limited by the type of somatic cells to be reprogrammed, that is, any type of somatic cell can be reprogrammed or dedifferentiated. Because reprogramming of a somatic cell does not require an ovule or / or an embryo, an iPS cell can be a mammalian cell, therefore, providing the opportunity to generate specific pluripotent stem cells for the patient or disease.

Viral, non-viral and non-integral methods for generating induced pluripotent stem cells (iPSC) using adenovirus, plasmids or cleavage of reprogramming factors using Cre-loxP3 or piggy BAC transposition have been described. See Stadtfeld, M., et al., Science 322, 945-949 (2008); Okita, K. et al., Science 322, 949-953 (2008); Kaji, K. et al. Nature 458, 771-775 (2009); Soldner, F. et al. Cell 136, 964-977 (2009); and Woltjen, K. et al. Nature 458, 766-770 (2009). Also, see US Pat. No. 20100003757 by Mack, A. et al. (published on January 7, 2010) and No.: PCT / US2009 / 037429 by Shi et al. These methods, however, have low reprogramming efficiencies (<0.003%), and may leave residual vector sequences despite cleavage, which limits their therapeutic applications. For example, viral integration in the host genome and overexpression of the above transcription factors have been associated with tumorigenesis; and a residual transgene expression is potentially the characteristic that distinguishes ES cells and iPS cells. See Solder, F. et al., Cell 136: 964-977 (2009); Foster et al., Oncogene 24: 1491-1500 (2005); and Hochedlinger, K. et al., Cell 121: 465-477 (2005).

In other embodiments of the invention, methods for generating iPSC include episomic vectors derived from the Epstein-Barr virus. See Yu, J. et al. Science 324, 797-801 (2009) and the US application. UU. 20100003757 de Mack, A. et al. published on January 7, 2010. These methods require three separate plasmids that carry a combination of seven factors, including the SV40 oncogene.

In another embodiment of the invention, methods for generating iPSC include iPSC based on human and mouse fetal and neonatal cell proteins. See Zhou H. et al. Cell Stem Cell 4, 381-384 (2009); and Kim, D. et al. Cell Stem Cell 4, 472-476 (2009). These methodologies are performed using a chemical treatment (for example, valproic acid in the case of Zhou et al. 2009 supra) or several treatment series (Kim et al. 2009, supra).

In another embodiment of the invention, mini-circle vectors or plasmids, which are supercoiled DNA molecules that lack an origin of bacterial replication and antibiotic resistance genes, can be used. See Chen Z.-Y. et al., Mol. Ther. 8, 495-500 (2003); Chen, Z.-Y. et al., Hum. Gene Ther. 16, 126-131 (2005); and Jia, F. et al., "Nature Methods Advance Publication" online February 7, 2010. These methodologies generate iPSC with abundant transfection efficiencies and longer ectopic expression, because they have lower activation of silencing mechanisms. exogenous

In yet another embodiment of the invention, iPS cells can be generated from human patients with various diseases, including diabetic patients, ALS patients, patients with spinal muscular dystrophy and Parkinson's. See Maehr et al. PNAS USA 106 (37): 15768-73 (2009); Dimos et al., Science, 321: 1218-21 (2008); Ebert et al. Nature 457: 277-80 (2009); Park et al. Cell 134: 877-886 (2008); and Soldner et al., Cell 136: 964-977. At least one advantage of producing hIPS cells from patients with specific diseases is that the derived cell would contain the genotype and cellular responses of the human disease. Also, see Table 4 that lists at least some strains of existing human iPS cells. This information was derived from published material and publicly available databases, including, for example, the Registry of Stem Cells of the National Institutes of Health (NIH), the Registry of Human Embryonic Stem Cells and the International Registry of Stem Cells located at the University of Massachusetts School of Medicine, Worcester, Massachusetts, USA. These databases are updated periodically as cell lines are available and registration is obtained.

The reference compositions and methods described herein contemplate the use of various differentiable primate pluripotent stem cells, including human pluripotent stem cells, such as hESC, including, but not limited to, CyT49, CyT212, CyT203, CyT25, (available on the market at least at the time of submission of the present application of ViaCyte Inc. located at 3550 General Atomics Court, San Diego CA 92121) BGO1, BG02 and m El 1, and induced pluripotent stem cells (iPS) such as iPSC- 482c7 and iPSC-603 (commercially available from Cellular Dynamics International, Inc., Madison, Wisconsin) and iPSC-G4 (hereafter "G4") and iPSC-B7 (hereafter "B7") ( commercially available at Shinya Yamanaka, Center for iPS Cell Research, University of Kyoto); Studies using these and other human iPS cells are described in detail in US patent applications. 12 / 765,714 and 13 / 761,078, both entitled "CELL COMPOSITIONS FROM DEDIFFERENTIATED REPROGRAMMED CELLS", filed on April 22, 2010 and February 6, 2013, respectively. Some of these human pluripotent stem cells are registered in national registries such as the National Institutes of Health (NIH) and are listed in the NIH Human Stem Cell Registry (for example, CyT49 registry No. 0041). You can also find information about CyT49, other cell lines available on the world website stemcells.nih.gov/research/registry. Other lines of cells, for example, BG01 and BG01v, are sold on the market and distributed to third parties by WiCell®, a subsidiary of the International Stem Cell Bank of Wisconsin (WISC) (catalog name, BG01) and ATCC ( Catalog No. SCRC-2002), respectively. While other cell lines described herein may not be registered or distributed by a biological repository, such as WiCell® or ATCC, such cell lines are commercially available to the general public directly or indirectly from those Main researchers, laboratories and / or institutions. Public applications for cell lines and reagents, for example, are common for experts in the field of life sciences. Generally, the transfer of these cells or materials is carried out by a conventional material transfer agreement between the owner of the cell line or the material and the recipient. These types of material transfers occur frequently in a research environment, particularly in the life sciences. In fact, the applicant has regularly transferred cells from the moment they were derived and characterized, including CyT49 (2006), CyT203 (2005), Cyt212 (2009), CyT25 (2002), BG01 (2001), BG02 ( 2001), BG03 (2001) and Bg 01v (2004), through agreements with business partners, and non-profit organizations and collaborators. The year in parentheses next to each cell line in the above list indicates the year in which cell lines or materials were made public and immortal (for example, cell banks were created) and, therefore, have not been Performed or required the destruction of another embryo since the establishment of these cell lines for the preparation of the compositions and the implementation of the methods described herein.

Tables 4 and 5 are non-exhaustive lists of certain iPSC and hESC, respectively, which are available worldwide for research and / or commercial purposes, and are suitable for use in the methods and compositions of the present disclosure. The information in Tables 3 and 4 was obtained from published material and databases available to the general public, which include: for example, the Registry of Human Stem Cells of the National Institutes of Health (NIH), the Registry of Human Embryonic Stem Cells and the International Registry of Stem Cells located at the University of Massachusetts School of Medicine, Worcester, Massachusetts, USA. These databases are updated periodically as cell lines are available and registration is obtained.

The human iPSC described herein (at least iPSC-603 and iPSC-482-c7) was provided by Cellular Dynamics International, Inc. (Madison, Wisconsin, USA).

T l 4: Li ir l l m r l ri n in i r l r h m n hIP

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Another advantage of using hIPS cells is that said hIPS cells would be a population of immunologically matched autologous cells; and the patient's specific cells would allow studying the origin and progression of the disease. Therefore, it is possible to understand the causes of a disease, which can provide information that leads to the development of prophylactic and therapeutic treatments for the disease.

Pluripotent human embryonic stem cells (hES) (reference examples)

Reference methods are disclosed for obtaining definitive endodermal cells and, ultimately, any type of cell derived from the endodermal lineage, including, but not limited to, upper digestive tract endoderm, pancreatic endoderm, endocrine progenitor / precursor cells and / or cells that express hormones from the pancreatic islet using human embryonic stem cells (hES) as a starting material. These hES cells can be cells that originate from the morula, embryonic internal cell mass or those obtained from embryonic gonadal ridges. Human embryonic stem cells can be maintained in culture in a pluripotent state without substantial differentiation using methods known in the art. Such methods are described, for example, in US Pat. No. 5,453,357; 5,670,372; 5,690,926; 5,843,780; 6,200,806 and 6,251,671.

In some processes, pluripotent stem cells, for example, hES cells, are maintained in a feeder layer. In such processes, any feeder layer that allows the pluripotent cell to be maintained in a pluripotent state can be used. A feeder layer commonly used for the culture of human embryonic stem cells is a layer of mouse fibroblasts. More recently, human fibroblast feeder layers have been developed for use in the cultivation of pluripotent cells (see US Patent Application Publication No. 2002/0072117). Alternative processes allow the maintenance of pluripotent cells without the use of a feeder layer.

The pluripotent cells described herein can be maintained in culture with or without serum, with or without extracellular matrix, with or without FGF. In some pluripotent cell maintenance procedures, serum replacement is used. These and other methods for the cultivation and differentiation of pluripotent or multipotent cells, respectively, are described in PCT / US2007 / 062755, filed on February 23, 2007, and entitled "COMPOSITIONS AND METHODS FOR CULTURING DIFFERENTIAL CELLS" and the document PCT / US2008 / 080516, filed on October 20, 2008, and entitled "METHODS AND COMPOSITIONS FOR FEEDER-FREE PLURIPOTENT STEM CELL MEDIA CONTAINING HUMAN SERUM".

The reference methods described herein are useful with all strains of hES cells, and at least those listed in Table 5, which are commercially available from the identified company or to be purchased on the Internet WiCell market at wicell.org/home/stem-cell-lines/order-stem-cell-lines/obtain-stemcell-lines.cmsx. This information was derived from published material and publicly available databases, including, for example, the Registry of Stem Cells of the National Institutes of Health (NIH), the Registry of Human Embryonic Stem Cells and the International Registry of Stem Cells located at the University of Massachusetts School of Medicine, Worcester, Massachusetts, USA. These databases are updated periodically as cell lines are available and registration is obtained. There are at least 254 strains of iPSC available in the market with the International Registry of Stem Cells and 1,211 strains of hESC available in the market. The following Table 5 does not include all the hESC and iPSC that are listed, but, rather, are examples of the pluripotent stem cells that are available.

Table 5: List of stretches of human embryonic stem cells hES

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Alternative methods for obtaining pluripotent stem cells

There are methods for obtaining pluripotent stem cells, such as mammalian ES cells, without the destruction of the embryo. In summary, Advanced Cell Technology (Worcester, Massachusetts, USA) published 3 articles in scientific journals describing the obtaining of mouse and human ES cells from individual blastomeres leaving the embryo intact and, therefore, without cause their destruction At the end of 2005, Chung et al. He first described methods for creating mouse ES cells from a single blastomer. See Chung et al. (2006) Nature 439: 216-219, published online Sunday, October 16, 2005. Chung et al. (2006) described the biopsy of an embryo using micromanipulation techniques similar to those used for preimplantation genetic diagnosis (PGD); see page 217. At that time, Chung et al. (2006) co-cultured blastomere lines with other embryonic stem cells, but subsequently developed methods in which this was not necessary. See Chung et al. (2008) "Human Embryonic Stem Cell Lines Generated without Embryo Destruction", Cell Stem Cell 2: 113-117.

The genetic diagnosis prior to implantation is used to analyze the genetic abnormalities of the embryos before implantation. The method is performed in normal developing embryos at an early stage (for example, the phase of the eight cells), when the genetic contribution of both parents can be studied, perforating the zona pellucida and aspirating or extruding or dissecting one or two blastomeres to Through the opening. Genetic analysis using fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) is what is commonly used in DGP to analyze chromosomal abnormalities and monogenic disorders, respectively. Subsequently, if there are no genetic abnormalities, the intact embryo is implanted in the patient for the normal gestation period. Genetic diagnostic techniques prior to implantation began to be published as of 2004 by several groups, including Staessen, C. et al. (2004), "Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial", Hum. Play 19: 2849-2858 and Monni et al. (2004) "reimplantation genetic diagnosis for beta-thalassaemia: the Sardinian experience". Prenat Diagn 24: 949-954. Therefore, Chung et al. (2006) limited themselves to describing the methods available to extract blastomeres from embryos in early stages without destroying the embryo.

In addition, in August 2006, Klimanskaya et al., The 2nd author of the publication of Chung et al. (2006) and also from Advanced Cell Technology, described a procedure, which although it was not effective (only 2% of the isolated blastomers generated a line of hES cells), managed to demonstrate that similar PGD techniques made it possible to obtain strains of human ES cells from a single blastomer; not significantly different from the one first described by Chung et al. (2006) supra. See Klimanskanya et al. (2006) "Human embryonic stem cell lines derived from single blastomeres", Nature 444, 481-485. Klimanskaya et al. "co-cultured the newly derived hES cell lines with other ES cells", as stated by Chung et al. (2006), may be fundamental. Chung et al. (2006) stated that "it is not clear whether the success of the joint ES culture system in (his) study is attributable to substances secreted by ES cells or if cell-cell contact is required." See Chung et al. (2006) supra, p. 218, right column. But a subsequent 2008 study by Chung themselves. et al., supra, demonstrated that hES cell lines did not require joint culture with ES cells at all, because the culture of blastomeres isolated in laminin media improved their ability to give rise to hESC. See Chung et al. (2008), "Human Embryonic Stem Cell Lines Generated without Embryo Destruction", Cell Stem Cell (2): 113-117, p. 116, published online on Thursday, January 10, 2008. In addition, that hES cells obtained from this way they had the same characteristics as other human pluripotent stem cells, including hES cells, being able to even maintain an undifferentiated state for more than six (6) months, and showing a normal karyotype and expression of pluripotency markers, including Oct -4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Nanog and alkaline phosphatase; and can differentiate and form derivatives of the three (3) embryonic germ layers both in vitro and in vivo teratomas .

Therefore, if the joint culture of individual blastomeres with other ES cells is not essential as Chung et al. (2006), and that cultivate them only in lamines, which is a common component of many extracellular matrices (commercially available and / or manufactured by lysis of feeder cells, for example), and was commonly used and known to cultivate mammalian cells , and such was known and available at the time of the first demonstration of obtaining ES mouse cell strains from a single blastomer according to Chung's publication in 2006. Therefore, it is completely possible for a person skilled in the art to be able to adopt the methodologies of Chung et al. (2006) supra and use the knowledge available to them as described above by Straessen et al. (2004) supra for obtaining a line of hES cells from a single blastomer while preserving the embryo or preventing its destruction.

Suspension of aggregates of pluripotent stem cells and cells derived from pluripotent stem cells

Unlike the previously known tissue engineering methods, which are based on seeding individual cells in polymeric frameworks, matrices and / or gels, the embodiments described herein may use suspensions of cell aggregates formed from pluripotent stem cells, individual cell suspensions or differentiated individual cell suspensions derived therefrom. The methods of processing and / or manufacturing the suspension of stem cell aggregates and the differentiation of the cells thereof are described in document PCT / US2007 / 062755, filed on April 23, 2007, and entitled "COMPOSITIONS AND METHODS FOR CULTURING DIFFERENTIAL CELLS ", and, now, US Pat. 8,211,699 and 8,415,158; and document PCT / US2008 / 080516, filed on October 2, 2008, and entitled "METHODS AND COMPOSITIONS FOR FEEDER-FREE PLURIPOTENT STEM CELL MEDIA CONTAINING HUMAN SERUM", and now US Pat. No. 8,334,138, and Schulz T. et al. (2012) supra.

The embodiments described herein refer to methods for generating an aggregate of pluripotent cells in suspension from a pluripotent adherent culture, culturing a pluripotent cell in a condition of adherent growth culture that allows expansion in an undifferentiated state; Dissociating the culture of adherent pluripotent cells in a suspension culture of individual cells; contacting the suspension culture of individual cells with a first differentiation culture condition that allows the formation of aggregates of cells derived from hES in suspension by stirring the suspension culture of individual cells for a period of time such that the suspension culture of individual cells form an aggregate of cells derived from pluripotent in suspension, and thus generating an aggregate of cells derived from pluripotent in suspension. In preferred embodiments, agitation of the suspension culture of individual cells is performed by rotation at about 80 rpm at 160 rpm. In certain other embodiments described herein, a rho-kinase inhibitor is used to facilitate aggregation, growth, proliferation, expansion and / or cell mass of pluripotent stem cells.

The term "substantially uniform" or "substantially uniform in size and shape" or its equivalents, refers to the spread in uniformity of the aggregates and is not greater than about 20%. In another embodiment, the uniformity propagation of the aggregates is not greater than about 15%, 10% or 5%.

In yet another embodiment, suspensions of hES cell aggregates were grown in a serum-free medium and, in addition, in the absence of exogenously added fibroblast growth factor (FGF). This is distinguished from the US patent. UU. No. 7,005,252 to Thomson, J., which requires culturing hES cells in a serum-free medium but containing exogenously added growth factors, including FGF. In some embodiments, suspensions of aggregates of iPS cells are cultured in a medium substantially free of serum and / or, in addition, in the absence of exogenously added fibroblast growth factor (FGF).

Although the exact number of cells per aggregate is irrelevant, those skilled in the art will recognize that the size of each aggregate (and, therefore, the number of cells per aggregate) is limited by the ability of oxygen and nutrients to diffuse into the central cells, and that this number can also vary depending on the type of cell and the nutritional needs of that type of cell. Cellular aggregates may comprise a minimum number of cells (for example, two or three cells) per aggregate, or they may comprise many hundreds or thousands of cells per aggregate. Typically, cell aggregates comprise hundreds to thousands of cells per aggregate. For the purposes of the present invention, cell aggregates are usually about 50 micrometers to about 600 micrometers in size, although, depending on the type of cell, the size may be less than or greater than this range. In one embodiment, the cell aggregates are about 50 micrometers to about 250 micrometers in size, or about 75 to 200 micrometers in size, and preferably have a size of about 100 to 150 micrometers.

Other methods describe the creation of embryoid bodies (EB). As used herein, the term "embryoid bodies", "aggregate bodies" or their equivalents, refers to aggregates of differentiated and undifferentiated cells that appear when ES cells grow excessively in monolayer cultures, or are maintained in cultures in suspension in indefinite media or are differentiated through protocols not directed towards tissues of multiple germ layers. That is, EBs are not formed from a suspension of individual pluripotent stem cell cells as disclosed herein; EB is also not formed from adherent cultures of multipotent cells derived from pluripotents. Only these characteristics make the present invention clearly distinguishable from an embryoid body.

In contrast to embryoid bodies, which are a mixture of differentiated and undifferentiated cells and that normally consist of cells of several germ layers and undergo random differentiation, the cellular aggregates described herein are essentially or substantially homocellular, existing as aggregates. of pluripotent, multipotent, bipotent or unipotent cells, for example, embryonic cells, definitive endoderm, upper digestive tract endoderm, PDX1 positive pancreatic endoderm, pancreatic endocrine cells and the like.

The methods described herein in no way require first coating the culture vessels. with an extracellular matrix, for example, as described in US Pat. No. 6,800,480 to Bodnar et al. and assigned to Geron Corporation. In some embodiments described herein, iPS cells can be cultured in the same manner as other pluripotent stem cells, for example, hES and iPS cells are cultured using soluble human serum as essentially described in US Pat. . No. 8,334,138 of the applicant; and Schulz T. et al. (2012) supra.

The methods described herein do not in any way require that fibroblast growth factor (FGF) supplied from a source other than just a fibroblast feeder layer as described in US Pat. 7,005,252 to Thomson, J. and assigned to the Wisconsin Alumni Research Foundation (WARF).

Multipotent and differentiated cell compositions

The cellular compositions produced by the methods described herein include cell cultures comprising pluripotent stem cells, in preprimitive, mesenddermic, definitive endodermal, upper digestive tract, upper digestive tract endodermal positive PDX1, pancreatic endodermal PDX1 positive or positive pancreatic endodermics in both PDX1 and NKX6.1, endocrine progenitors / precursors / endocrine progenitors / precursors positive in both NGN3 and NKX2.2 and hormone secreting endocrine cells or INS, GCG, GHRL, SST, positive endocrine cells only in PP, in which at least about 5-90% of the cells in culture are cells in preprimitive, mesedermal, definitive endodermal, upper digestive tract, upper digestive tract endodermal positive PDX1, P-positive pancreatic endodermal DX1 or positive pancreatic endodermics in both PDX1 and NKX6.1, endocrine progenitors / precursors or endocrine progenitors / precursors positive in both NGN3 and NKX2.2 and hormone secreting endocrine cells or positive endocrine cells only in INS, GCG, GHRL, SST, Pp produced.

Some embodiments described herein relate to compositions, such as cell populations and cell cultures comprising both pluripotent cells, such as stem cells and iPS cells, as multipotent cells, such as cells in preprimitive, mesedermal or enddermal striae, as well. as more differentiated cells, but still potentially multipotent, such as PDX1 positive upper endodermic cells, PDX1 positive pancreatic endodermal or PDX1 or NKX6.1 positive pancreatic endodermal cells, endocrine progenitors / precursors or endocrine progenitors / precursors in both NGN3 and NKX2.2 and endocrine hormone secreting cells or positive endocrine cells only in INS, GCG, GHRL, SST, PP. For example, using the methods described herein, compositions comprising mixtures of pluripotent stem cells and other multipotent or differentiated cells can be produced. In some embodiments, compositions are produced comprising at least about 5 multipotent or differentiated cells for about every 95 pluripotent cells. In other embodiments, compositions are produced comprising at least about 95 multipotent or differentiated cells by about every 5 pluripotent cells.

In addition, compositions comprising other proportions of multipotent or differentiated cells with respect to pluripotent cells are contemplated. For example, compositions comprising at least about 1 multipotent or differentiated cell for approximately every 1,000,000 pluripotent cells, at least about 1 multipotent or differentiated cell per 100,000 pluripotent cells, at least about 1 multipotent or differentiated cell for each 10,000 pluripotent cells, at least approximately 1 multipotent or differentiated cell per 1000 pluripotent cells, at least approximately 1 multipotent or differentiated cell per 500 pluripotent cells, at least approximately

1 multipotent or differentiated cell for every 100 pluripotent cells, at least about 1 multipotent or differentiated cell for every 10 pluripotent cells, at least about 1 multipotent or differentiated cell by about every 5 pluripotent cells, and up to about 1 pluripotent cell and at least about 1,000,000 multipotent or differentiated cells by approximately 1 pluripotent cell.

Some embodiments described herein refer to cell cultures or cell populations comprising at least about 5% multipotent or differentiated cells to at least about 99% multipotent or differentiated cells. In some embodiments, cell cultures or cell populations comprise mammalian cells. In preferred embodiments, cell cultures or cell populations comprise human cells. For example, certain specific embodiments relate to cell cultures comprising human cells, in which from at least about 5% to at least about 99% of human cells are multipotent or differentiated cells. Other embodiments relate to cell cultures comprising human cells, in which at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at minus about 65 about 70%, at least about 75%, at least about 80% about 85%, at least about 90%, at least about 95% approximately 98%, at least approximately 99 % or more than 99 % of human cells are multipotent or differentiated cells. In embodiments in which cell cultures or cell populations comprise human feeder cells, the above percentages are calculated with respect to human feeder cells in cell cultures.

The compositions and methods described herein have several useful features. For example, cell cultures and cell populations comprising multipotent cells, for example, cells in preprimitive striae and / or mesendodermal cells, as well as the methods for producing such cell cultures and cell populations, are useful for modeling the early stages of development. human. Likewise, the compositions and methods described herein can also be used for therapeutic intervention in pathological conditions, such as diabetes mellitus. For example, since cells in preprimitive stretch marks and / or mesendodermal cells serve as a source for only a limited number of tissues, they can be used in the development of tissues or pure cell types. In some production processes of preprimitic stria cells, the pluripotent cells used as starting material are pluripotent stem cells, for example, hES, hEG or iPS cells.

Troofectodermal cells

Using the methods described herein, compositions comprising trofectodermal cells essentially free of other cell types can be produced. In some embodiments described herein, the troofectodermal cell populations or cell cultures produced by the methods described herein have a high expression of markers selected from the group comprising HAND1, Eomes, MASH2, ESXL1, HCG, KRT18, PSG3, SFXN5, DLX3, PSX1, ETS2 and ERBB compared to the expression levels of HAND1, Eomes, MASH2, ESXL1, HCG, KRT18, PSG3, SFXN5, DLX3, PSX1, ETS2 and ERBB in non-trophorethermal cells or populations of cells .

Cells in preprimitive stretch marks

Using the methods described herein, compositions comprising cells in preprimitive stretch marks substantially free of other cell types can be produced. In some embodiments described herein, cell populations in preprimitive stretch marks or cell cultures produced by the methods described herein essentially express FGF8 and / or NODAL marker genes compared to a low level of BRAQUIURIA, low level of FGF4, low level of SNAI1, low level of SOX17, low level of FOXA2, low level of SOX7 and low level of SOX1. Cells in preprimitive stretch marks and methods for producing cells in preprimitive stretch marks are described in detail in US Pat. UU. of applicant No. 7,958,585, "PREPRIMITIVE STREAK AND MESENDODERM CELLS", granted on Tuesday, July 26, 2011.

Extraembryonic cells

Using the methods described herein, compositions comprising extraembryonic cells substantially free of other cell types can be produced. Primitive, visceral and parietal endoderm cells are extraembryonic cells. Primitive endoderm cells give rise to visceral and parietal endoderm cells. Visceral endoderm cells are endodermal cells that are part of the yolk sac. Visceral endoderm cells function in both absorption and nutrient transport. The cells of the parietal endoderm are contiguous to an extraembryonic tissue known as the Reichert membrane. One of the functions of parietal endoderm cells is to produce components of the basement membrane. Together, visceral endoderm cells and parietal endoderm cells form what is often called extraembryonic endoderm. As the name suggests, extra-embryonic endodermal cells do not give rise to embryonic structures formed during development. On the contrary, the definitive endodermal cells and other endodermal or pancreatic lineage cells described herein are embryonic or derived from embryonic cells and give rise to tissues that derive from the digestive tract that is formed during embryonic development. In some embodiments described herein, extraembryonic cell populations or cell cultures produced by the methods described herein have a high expression of markers selected from the group comprising the SOX7, SOX17, THBD, SPARC, DAB1 genes, HNF4alpha or AFP compared to the expression levels of at least SOX7, SOX17, THBD, SPARC, DAB1 or AFP, which is not expressed in other cell types or cell populations, for example, definitive endoderm.

Mesedermal cells

Using the methods described herein, compositions comprising mesedermal cells substantially free of other cell types can be produced. In some embodiments described herein, populations of mesedermal cells or cell cultures produced by the methods described herein have a high expression of markers selected from the group comprising the FGF4, SNAI1 MIXL1 and / or WNT3 genes, in comparison with low level of SOX17, low level of CXCR4, low level of FOXA2, low level of SOX7 and low level of SOX1. The mesenddermal cells and the methods to produce mesenddermal cells are described in detail in US Pat. UU. of applicant No. 7,958,585, "PREPRIMITIVE STREAK AND MESENDODERM CELLS", granted on Tuesday, July 26, 2011.

Systematic Screening Method

In some embodiments, systematic screening methods are employed to obtain certain cell populations comprising multipotent and / or differentiated, pluripotent cells, such as human pluripotent stem cells, induced pluripotent stem cells, cells sown in preprimitive striae, mesedermal cells, definitive endodermal cells , endodermal cells of the upper digestive tract or endodermal cells of the upper digestive tract negative in PDX1, endodermal cells of the upper digestive tract positive in PDX1 or pancreatic positive endodermal cells in PDX1 or pancreatic progenitor cells, endocrine progenitor / precursor cells and / or endocrine cells. The cell population is then given a candidate differentiation factor. At a first time point, which is earlier or approximately equal to the time at which the candidate differentiation factor is provided, the expression of a marker is determined. Alternatively, the expression of the marker can be determined after providing the candidate differentiation factor. At a second time point, which is after the first time point and after the stage of providing the candidate differentiation factor to the cell population, the expression of the same marker is determined again. It is determined whether the candidate differentiation factor is capable of enhancing the differentiation of pancreatic precursor cells by comparing the expression of the marker at the first time point with the expression of the marker at the second time point. If the expression of the marker at the second time point increases or decreases compared to the expression of the marker at the first time point, then, the candidate differentiation factor is able to enhance the differentiation of pancreatic progenitor cells.

Some embodiments of the systematic screening methods described herein utilize cell populations or cell cultures that comprise human definitive endodermal cells, negative upper endodermal cells in PDX-1, upper digestive cells of the upper digestive tract in PDX-1, PDX-1 pancreatic endodermal cells or pancreatic progenitor or endocrine progenitor / precursor cells. For example, the cell population may be an essentially purified population of pancreatic endodermal cells positive in PDX-1-or pancreatic progenitor cells. For example, the cell population may be an enriched population of human pancreatic progenitor cells, in which at least about 50% to 97% of the human cells of the cell population are human pancreatic progenitor cells, the rest comprising progenitor / precursor cells. endocrine or endocrine cells and other cell types. The enrichment of pancreatic progenitor populations is described in detail in the US patent application. UU. No. 12 / 107,020 of the applicant, entitled "METHOD FOR PURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS", filed on April 21, 2008, now US Pat. of 8,338,170 and the corresponding publication of Kelly et al. (2011) supra.

In embodiments of the systematic screening methods described herein, the cell population is contacted or otherwise receives a candidate differentiation factor (test). The candidate differentiation factor may comprise any molecule that may have the potential to promote the differentiation of any of the aforementioned cells, for example, human pancreatic progenitor cells. In some embodiments described herein, the candidate differentiation factor comprises a molecule that is known to be a differentiation factor for one or more cell types. In alternative embodiments, the candidate differentiation factor comprises a molecule that is not known to enhance cell differentiation. In preferred embodiments, the candidate differentiation factor comprises a molecule that is not known to enhance the differentiation of human pancreatic progenitor cells. Systematic screening of factors that differentiate the final endoderm, for example, is described in detail in the US patent application. No. 12 / 093,590, entitled "MARKERS OF DEFINITIVE ENDODERM", filed on July 21, 2008.

In addition to determining the expression of at least one marker at a first time point, some embodiments of the systematic screening methods described herein contemplate determining the expression of at least one marker at a second time point that is subsequent to the first point of time and that is later to provide the cell population with the candidate differentiation factor. In said embodiments, the expression of the same marker in the first and second time points is determined. In some embodiments, the expression of a plurality of markers in the first and second time points is determined. In said embodiments, the expression of the same plurality of markers in the first and second time points is determined. In some embodiments, marker expression is determined at a plurality of time points, each of which is subsequent to the first time point, and each of which is subsequent to providing the cell population with the candidate differentiation factor. . In certain embodiments, Marker expression is determined by Q-PCR. In other embodiments, marker expression is determined by immunocytochemistry.

In certain embodiments of the systematic screening methods described herein, the marker having its expression determined in the first and second time points is a marker that is associated with the differentiation of pancreatic progenitor cells to cells that are the precursors of terminal differentiated cells that form the tissues of the pancreatic islets. Such cells may include immature cells that express the hormone of the pancreatic islet.

In some embodiments of the systematic screening methods described herein, sufficient time is allowed between the supply to the cell population of the candidate differentiation factor and the determination of the expression of the marker in the second time point. The sufficient time between the supply to the cell population of the candidate differentiation factor and the determination of the expression of the marker at the second time point can be as short as about 1 hour to about 10 days. In some embodiments, the expression of at least one marker is determined several times after providing the candidate population with the differentiation factor. In some embodiments, sufficient time is at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 16 hours, at least about 1 day, at least about 2 days, at least about 3 days , at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at at least about 12 days, at least about 13 days, at least about 14 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks or at least about 8 weeks.

In some embodiments of the methods described herein, it is also determined whether the expression of the marker at the second time point has increased or decreased compared to the expression of this marker at the first time point. An increase or decrease in the expression of at least one marker indicates that the candidate differentiation factor is capable of enhancing the differentiation of endocrine progenitor / precursor cells. Similarly, if the expression of a plurality of markers is determined, it is further determined whether the expression of the plurality of markers at the second time point has increased or decreased compared to the expression of this plurality of markers at the first point. of time. An increase or decrease in marker expression can be determined by measuring or evaluating the amount, level or activity of the marker in the cell population at the first and second time points. Such determination may be relative to other markers, for example, the expression of constitutive genes, or absolute. In certain embodiments, in which the expression of the marker increases at the second time point compared to the first time point, the amount of increase is at least about 2 times, at least about 5 times, at least about 10 times , at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at less about 100 times or more than about 100 times. In some embodiments, the amount of increase is less than 2 times. In embodiments where the expression of the marker decreases at the second time point compared to the first time point, the amount of decrease is at least about 2 times, at least about 5 times, at least about 10 times, at at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times or more than about 100 times. In some embodiments, the amount of decrease is less than 2 times.

Control of the production of multipotent or differentiated cells

The progression of pluripotent cells to multipotent cells to multipotent cells or additional differentiated cells, such as pancreatic progenitor cells or hormonal endocrine secreting cells, can be controlled by determining the expression of characteristic markers of specific cells, including genetic markers and phenotypic markers, such as Islet hormone expression and proinsulin processing in insulin and C-peptide in endocrine cells. In some processes, the expression of certain markers is determined by detecting the presence or absence of the marker. Alternatively, the expression of certain markers can be determined by measuring the level at which the marker is present in the cells of the cell culture or the cell population. For example, in certain processes, the expression of characteristic markers of immature cells expressing pancreatic islet hormones is determined, as well as the lack of significant expression of characteristic markers of pluripotent cells, definitive endodermal, endodermal of the upper digestive tract, endodermal of the tract upper digestive positive in PDX1, endocrine progenitors / precursors, extra-embryonic, mesodermal, ectodermal, mature cells expressing pancreatic islet hormones and / or other cell types.

As described in connection with the control of the production of other types of cells less differentiated from the definitive endodermal lineage, qualitative or semi-quantitative techniques, such as point transfer and immunocytochemical methods, can be used to measure marker expression. Alternatively, the expression of the marker can be quantified accurately by using a technique such as Q-PCR or nCounter using Nanostring or related high-performance multiplex technologies, capable of analyzing and evaluating hundreds or more markers simultaneously. In addition, it will be appreciated that, at the polypeptide level, many of the markers of cells expressing pancreatic islet hormones are secreted proteins. As such, techniques can be used to measure the content of extracellular markers, such as ELISA.

In other embodiments, immunohistochemistry is used to detect proteins expressed by genes. mentioned above. In yet other embodiments, the Q-PCR may be used in conjunction with immunohistochemical techniques or flow cytometry techniques to accurately and accurately characterize and identify cell types and determine both the amount and relative proportions of said markers in one type. of cell under study. In one embodiment, the Q-PCR can quantify the levels of RNA expression in a cell culture that contains a mixed population of cells. However, Q-PCR cannot provide or qualify if the subject's markers or proteins are expressed simultaneously in the same cell. In another embodiment, the Q-PCR is used in conjunction with flow cytometry methods to characterize and identify cell types. Therefore, by using a combination of the methods described herein, and as described above, the characterization and complete identification of various cell types, including endodermal lineage type cells can be performed and demonstrated.

For example, in a preferred embodiment, pancreatic or pancreatic or pancreatic endodermic progenitor cells in PDX-1 positive, express at least PDX1, Nkx6.1, PTF1A, CPA and / or cMYC as demonstrated by Q-PCR and / or ICC, but said cell jointly expresses at least PDX1 and Nkx6.1 as demonstrated by ICC and does not express other markers, including SOX17 CXCR4 or CER, to be identified as a cell expressing PDX1-positive. Similarly, for the proper identification of a mature hormone secreting pancreatic cell, in vitro or in vivo, for example, it has been shown that C-peptide (a product of the proper processing of proinsulin in a mature and functional p cell) and insulin are expressed together by ICC in the insulin secretory cell.

Even so, other methods known in the art can also be used to quantify marker gene expression. For example, the expression of a marker genetic product can be detected using antibodies specific for the marker gene product of interest (eg, Western blot, flow cytometric analysis and the like). In certain processes, the expression of the characteristic marker genes of the cells derived from hES, as well as the lack of significant expression of the characteristic marker genes of the cells derived from hES. Other additional methods for characterizing and identifying hES derived cell types are described in related applications as indicated above.

Summary of production of positive pancreatic endodermal cells in PDX1 (Phases 1 to 4) and insulin production in vivo

Methods for the production of certain cells of the endodermal lineage and cells of the pancreatic endodermal lineage are provided herein, and are discussed elsewhere in related applications such as US Pat. No. 7,534,608; 7,695,965; and 7,993,920, entitled "METHODS OF PRODUCING PANCREATIC HORMONES"; and U.S. Pat. No. 8,129,182, entitled "ENDOCRINE PROGENITOR / PRECURSOR CELLS, PANCREATIC HORMONE-EXPRESSING CELLS AND METHODS OF PRODUCTION". See also Table 17 and FIG. 42, 43 and 44.

In summary, the methods of directed differentiation herein for pluripotent stem cells, for example, hES and iPS cells, can be described in at least four or five or six or seven phases, depending on the final phase of the desired cell culture (PEC or endocrine cells). Phase 1 is the production of definitive endodermal cells from pluripotent stem cells and takes approximately 2 to 5 days, preferably 2 or 3 days. Pluripotent stem cells are suspended in media comprising RPMI, a growth factor of members of the TGFp superfamily, such as Activin A, Activin B, GDF-8 or GDF-11 (100 ng / ml), a member of the Wnt family or a Wnt pathway activator, such as Wnt3a (25 ng / ml), and alternatively, a rhokinase inhibitor or ROCK, such as Y-27632 (10 jM) to enhance growth and / or survival and / or proliferation, and / or adhesion between cells. After about 24 hours, the media are exchanged for media comprising RPMI with serum, such as 0.2% FBS and a growth factor of TGFp superfamily members, such as Activin A, Activin B, GDF-8 or GDF-11 (100 ng / ml) and, alternatively, a rho-kinase inhibitor or ROCK for another 24 (day 1) to 48 hours (day 2). Alternatively, after approximately 24 hours in a medium comprising Activin / Wnt3a, the cells are cultured for the next 24 hours in a medium comprising Activin alone (ie, the medium does not include Wnt3a). Importantly, the production of definitive endoderm requires conditions of cell culture low in serum content and, therefore, low in insulin or in insulin-like growth factor content. See McLean et al. (2007) Stem Cells 25: 29-38. McLean et al. they also show that contacting the hES cells with insulin at concentrations as low as 0.2 jg / ml in Phase 1 can be detrimental to the production of the definitive endoderm. Other experts in the field have modified the differentiation of Phase 1 from pluripotent cells to definitive endodermal cells essentially as described herein and in D'Amour et al. (2005), for example, at least, Agarwal et al., "Efficient Differentiation of Functional Hepatocytes from Human Embryonic Stem Cells", Stem Cells (2008) 26: 1117-1127; Borowiak et al., "Small Molecules Efficiently Direct Endodermal Differentiation of Mouse and Human Embryonic Stem Cells", (2009) Cell Stem Cell 4: 348-358; and Brunner et al., "Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver", (2009) Genome Res.

19: 1044-1056. Appropriate differentiation, specification, characterization and identification of the definitive cells are necessary to obtain other cells of the endodermal lineage. The final endodermal cells in this phase jointly express SOX17 and HNF3p (FOXA2), and do not appreciably express at least HNF4alpha, HNF6, PDX1, SOX6, PROX1, PTF1A, CPA, cMYC, NKX6.1, NGN3, PAX3, ARX, NKX2. 2, INS, GSC, GHRL, SST or PP. The absence HNF4alfa expression in the definitive endoderm is supported and described in detail at least in Duncan et al. (1994), "Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo: HNF-4 is a marker for primary endoderm in the implanting blastocyst", Proc. Natl. Acad. Sci, 91: 7598-7602 and Si-Tayeb et al. (2010), "Highly Efficient Generation of Human Hepatocyte-Like cells from Induced Pluripotent Stem Cells", Hepatology 51: 297-305.

Phase 2 takes the culture of final endodermic cells of Phase 1 and produces endoderm of the upper digestive tract or endoderm of the upper digestive tract negative in PDX1 by incubating suspension cultures with RPMI with low serum levels, such as 0.2% FBS , in a 1: 1,000 dilution of STIs, 25 ng KGF (or FGF7), and alternatively, a ROCK inhibitor for 24 hours (from day 2 to day 3). After 24 hours (from day 3 to day 4), the media is exchanged by the same means minus a TGFp inhibitor, but, alternatively, it remains a ROCK inhibitor to enhance the growth, survival and proliferation of the cells, for another 24 (days 4 to 5) to 48 hours (day 6). A fundamental stage for the proper specification of the upper digestive tract endoderm is the elimination of the growth factors of the TGFp family. Accordingly, a TGFp inhibitor can be added to Phase 2 cell cultures, such as the 2.5 pM TGFp inhibitor # 4 or SB4315425 pM, a kinase specific inhibitor similar to the Activin receptor ( ALK), which is a type I TGFp receptor. The endodermal cells of the upper digestive tract or endodermal upper digestive tract negative in PDX1 produced from Phase 2 together express SOX17, HNF1p and HNF4alfa and do not expressly express at least SOX17 and HNF3p (FOXA2), nor HNF6, PDX1, SOX6 , PROX1, PTF1A, CPA, cMYC, NKX6.1, NGN3, PAX3, ARX, NKX2.2, INS, GSC, GHRL, SST or PP, which are the hallmark of definitive endodermal cells, positive pancreatic endodermal cells in PDX1 or pancreatic progenitor or endocrine progenitor / precursor cells, as well as normally, polyhormonal type cells.

Phase 3 (days 5-8) for the production of PEC takes the culture of upper digestive tract endodermal cells from Phase 2 and produces a positive upper digestive tract endodermal cell in PDX1 by DMEM or RPMI in 1% B27, 0.25 pM KAAD cyclopamine, a retinoid, such as 0.2 pM retinoic acid (RA) or a retinoic acid analog such as 3 nM TTNPB (or CTT3, which is the combination of KAAD and TTNPB cyclopamine) , and 50 ng / ml of Nogina for approximately 24 (day 7) to 48 hours (day 8). Specifically, Applicants have used DMEM with high glucose content since approximately 2003, and all patent and non-patent disclosures up to that time employed DMEM with high glucose content, even if it is not mentioned as "DMEM with high level of glucose "and the like. This is, in part, because manufacturers such as Gibco did not name their DMEM as such, for example, DMEM (cat. # 11960) and d Me M with deactivation (cat. # 10829). It should be noted that as of the date of submission of this application, Gibco offers more DMEM products, but still does not put "high glucose" in some of its DMEM products that contain high glucose, for example, a DMEM with deactivation (cat. # 10829-018). Therefore, it can be assumed that, in each case, in which DMEM is described, it means DMEM with high glucose content, and this was evident by others who are conducting research and development in this field. In Example 21, more details are described that describe the use of high exogenous glucose content. Again, a ROCK inhibitor or a rho-kinase inhibitor such as Y-27632 can be used to enhance growth, survival, proliferation and enhancement of cell-cell adhesion. PDX1 positive upper digestive tract cells produced from Phase 3 jointly express PDX1 and HNF6, as well as SOX9 and PROX, and do not appreciably express markers indicative of definitive endodermal cells or upper digestive tract endodermal cells (cells endodermal upper digestive tract negative in PDX1) or endodermal upper digestive tract cells positive in PDX1 as described above in Phases 1 and 2.

The method of Phase 3 above is that of four phases for the production of PEC. For the production of endocrine and endocrine progenitor / precursor cells as described in detail in Examples 9-24 below, in addition to Nogina, KAAD and Retinoid cyclopamine are used; Activin, Wnt and Heregulin, alone and / or combined, to suppress NGN3 expression while maintaining a good mass of cell aggregates. See Examples 8, 9 and 10.

The production of PEC of Phase 4 (days 8-14) takes the means of Phase 3 and exchanges them for means containing DMEM in a B27 supplement at 1% vol / vol, plus 50 ng / ml of KGF and 50 ng / ml of EGF and sometimes also Nogina at 50 ng / ml and a ROCK inhibitor, and also includes Activin alone or in combination with Heregulin. These new methods give rise to pancreatic progenitor cells that jointly express at least PDX1 and NKX6.1, as well as PTF1A. These cells do not appreciably express markers indicative of definitive endodermal cells or upper digestive tract endodermal cells (negative upper digestive tract endodermal in PDX1) as described above in Phases 1,2 and 3. See FIG. 44.

Alternatively, Phase 4 cells can be further differentiated in Phase 5 to produce endocrine progenitor or progenitor / precursor cells and / or pancreatic endocrine cells of a single hormone or of several hormones in a medium containing DMEM with supplement of B27 at 1% vol / vol for about 1 to 6 days (preferably about 2 days, that is, days 13-15). Endocrine progenitors / precursors produced from Phase 5 jointly express at least CHGA, NGN3 and Nkx2.2, and do not appreciably express the markers indicative of definitive endoderm or upper digestive tract endoderm (upper digestive tract endoderm negative in PDX1) as described above in Phases 1, 2, 3 and 4 for the PEC production. See FIG. 44.

For the production of PEC, PDX1-positive pancreatic endodermal cells produced in Phase 4 are fully loaded and introduced into a macroencapsulation device, and transplanted into a patient, and PDX1-positive pancreatic endodermal cells mature to secretory cells of pancreatic hormones or pancreatic islets, for example, insulin-secreting beta cells, in vivo (also known as " in vivo function"). Encapsulation of pancreatic endodermal cells positive in PDX1 and insulin production in vivo are described in detail in the US application. UU. No. 12 / 618,659 (Application 659), entitled "ENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS", filed on Friday, November 13, 2009. Application 659 claims the priority of the provisional patent application n. ° 61 / 114,857, entitled "ENCAPSULATION OF PANCREATIC PROGENITORS DERIVED FROM HES CELLS", presented on Friday, November 14, 2008; and the US provisional patent application No. 61 / 121,084, entitled "ENCAPSULATION OF PANCREATIC ENDODERM CELLS", presented on Tuesday, December 9, 2008; and now US Pat. No. 8,278,106 and 8,424,928. The methods, compositions and devices described herein are currently representative of preferred embodiments, and are illustrative and are not intended to be limitations on the scope of the invention. Experts in the field will come up with changes and other uses that are defined by the scope of the disclosure. Accordingly, it will be apparent to one skilled in the art that variable substitutions and modifications of the invention disclosed herein may be made.

For example, Activin A, a member of the TGFp superfamily of growth factors or signaling proteins, is used to produce definitive endoderm from pluripotent cells, for example, hES cells and iPS cells; however, other members of the TGFp superfamily, for example, GDF-8 and GDF-11, can be used to produce a definitive endoderm such as those described in international application PCT / US2008 / 065686, entitled "GROWTH FACTORS FOR PRODUCTION OF DEFINITIVE ENDODERM ", presented on Tuesday, June 3, 2008.

Still in a different context, Activin alone or in combination with Wnt and Heregulin is able to suppress and inhibit the expression of NGN3 at high and low levels; at low levels alone or in combination with Heregulin, or as an alternative, at high levels in combination with w Nt and Heregulin, cell mass and, therefore, performance can be maintained. See Examples 8, 9 and 10.

For the production of PEC, retinoic acid (RA) is used to differentiate negative upper digestive tract endodermal cells in PDX1 in Phase 2 to upper digestive tract cells positive in PDX1 in Phase 3. However, they can be used other retinoids or retinoic acid analogs such as 4 - [(E) -2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1-propenyl] benzoic (or TTNPB) and similar analogs (for example, 4-HBT-TNPB). For the production of endocrine progenitor / precursor cells and endocrine cells, retinoic acid or any of its analogs can also be added during Phases 6 and / or 7 to induce gene expression of hormones. See Examples 13 and 16.

The nogina is a protein that, for example, inactivates the members of TGFp superfamily signaling proteins, such as bone morphogenetic protein 4 (BMP4). However, other BMP4 inhibitors such as Cordine and Twisted Gastrulation (Tsg) or anti-BMP neutralizing antibodies can prevent BMP binding to their cell surface receptors, thus effectively inhibiting BMP signaling. Alternatively, the gene for human Nogin has been cloned and sequenced. See US Pat. No. 6,075,007. Nogina sequence analysis shows a carboxy terminal region that has homology with a Kunitz-like protease inhibitor, indicating that potentially other Kunitz-like protease inhibitors may have a similar effect on BMP inhibition. Example 15 describes the use of Nogina to increase the production of non-endocrine subpopulations (CHGA-).

Finally, the macroencapsulation devices described in this document and in Application 659; US design requests 29 / 447,944; 29 / 408,366; 29 / 408,368; 29 / 423,365; and 61 / 774,443, entitled "SEMIPERMEABLE MACRO IMPLANTABLE CELLULAR ENCAPSULATION DEVICES", presented on Thursday, March 7, 2013, again, are only examples, and are not intended to be limitations on the scope of the invention. In particular, all changes in the design of the device, such as the size of the device, the plurality of cameras or sub-compartments in the device, or the plurality of ports, or even the mechanisms for loading and removing the device are encompassed. Accordingly, it will be apparent to one skilled in the art that variable substitutions and modifications can be made not only of the differentiation methods described herein, but also of the encapsulation device, in the invention disclosed herein. See Examples 10, 12 and 18.

With respect to some of the processes for the differentiation of pluripotent cells to definitive endodermal cells, the growth factors mentioned above are provided to the cells so that the growth factors are present in the cultures at sufficient concentrations to enhance the differentiation of at least a part of pluripotent cells to definitive endodermal cells and pancreatic lineage cells. In some processes, the growth factors mentioned above are present in the cell culture at a concentration of at least about 5 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, at least about 1,000 ng / ml, at least about 2,000 ng / ml, at least about 3,000 ng / ml, at least about 4,000 ng / ml, at least about 5,000 ng / ml or more than about 5,000 ng / ml. Other agents or growth factors are present in cell cultures at a concentration of at least 0.1 mM or 10 mM, or more.

In certain processes, pluripotent stem cell differentiation agents and / or growth factors are removed from the cell culture after their addition. For example, Growth factors can be eliminated in approximately one day, approximately two days, approximately three days, approximately four days, approximately five days, approximately six days, approximately seven days, approximately eight days, approximately nine days or approximately ten days after Your addition In a preferred process, growth factors are removed approximately four days after their addition. The removal of the agent and / or the growth factor can be performed by changing the media in the absence of the agent and / or growth factor, or using another agent that inhibits the function of that agent and / or growth factor.

In a preferred embodiment, cell cultures in Phase 3 include, but are not limited to, agents capable of repressing, suppressing, inhibiting and the like, cells compromised with the endocrine lineage (CHGA +). Such agents also include Activin, Heregulin and WNT, and combinations of the three in amounts effective to enhance differentiation and / or induce expression of markers that are indicative of the subpopulation of non-endocrine multipotent pancreatic progenitor cells (CHGA-) and repress or minimize the expression of markers of cells compromised with the endocrine lineage (CHGA +). Indicative markers of cells committed to the endocrine lineage (CHGA +) include, but are not limited to, NGN3, NKX2.2 and others.

In another preferred embodiment, cell cultures in Phase 4 include, but are not limited to, agents capable of repressing, suppressing, inhibiting and the like, cells compromised with the endocrine lineage (CHGA +). Such agents also include Activin and Heregulin and combinations of the two in amounts effective to enhance differentiation and / or induce expression of markers that are indicative of a subpopulation of non-endocrine multipotent pancreatic progenitor cells (CHGA-) and repress or minimize expression of cell markers compromised with the endocrine lineage (CHGA +). Indicative markers of cells committed to the endocrine lineage (CHGA +) include, but are not limited to, NGN3, NKX2.2 and others. The means for suppressing at least NGN3 during phases 3 and 4 are described in detail in the following Examples 8-21.

In one embodiment, phase 4 cell cultures are further differentiated in phase 5 with at least Nogin, KGF, EGF and a signaling inhibitor or Notch pathway. Markers indicative of endocrine differentiation include, but are not limited to, NGN3, NKX2.2 and others. In a preferred embodiment, the agents are used in phases 1 7 that effectively simulate or mimic in vitro what is observed in in vivo development studies . For example, phases 3 and 4 of this document and in accordance with Table 17, use agents that are capable of delaying, repressing, suppressing and / or inhibiting endocrine differentiation, or delaying, repressing, suppressing and / or inhibiting indicative markers. of endocrine differentiation, including, but not limited to, NGN3 and NKX2.2. During phases 5, 6 and / or 7, cell cultures are treated with agents that are capable of inducing, increasing and / or enhancing endocrine differentiation, for example, by using a Notch pathway inhibitor, such as a gamma secretase inhibitor. In short phases, the cell culture conditions of phases 3 and 4 suppress endocrine phenotypes, while cell culture conditions of phases 5, 6 and / or 7 progressively induce endocrine phenotypes that include, but are not limited to, insulin ( INS), glucagon (GCG), somatostatin (SST), pancreatic polypeptide (PP), ghrelin (GHRL), member 8 of the 30 family of solute vehicles (SLC30A8), Glucose-6-phosphatase 2 (G6PC2), prohormone convertase 1 (PCSK1) and glucose kinase (GCK).

In one embodiment, PDX1 positive pancreatic endodermal cells are differentiated from endocrine and endocrine progenitor / precursor cells by continuing incubation of PDX1 positive pancreatic endodermal cells in the presence of a Notch signaling inhibitor, for example, an inhibitor of Notch signaling. gamma secretase such as R04929097, used alone or in combination with retinoids such as retinoic acid. The presence of a Notch signaling inhibitor induces the expression of NGN3 during phase 5. In other embodiments, the gamma secretase inhibitor is provided at the beginning of the differentiation process, for example, in the pluripotent phase, and remains in the cell culture throughout the differentiation to cells that express hormones of the pancreatic islet. In yet other embodiments, the gamma secretase inhibitor is added after the onset of differentiation, but before differentiation to the endoderm phase of upper positive digestive tract in PDX1. In preferred embodiments, the gamma secretase inhibitor is provided to the cell culture or cell population at approximately the same time as the differentiation factors that enhance the conversion of the definitive endoderm into positive endoderm in PDX1 are provided. In other preferred embodiments, the gamma secretase inhibitor is provided to the cell culture or the cell population after a substantial part of the cells of the cell culture or cell population has differentiated to upper digestive tract endodermal cells positive in PDX1. .

In another embodiment, Phase 5 cell cultures differ further in phases 6 and 7. During these phases, Agents that are capable of properly specifying endocrine progenitor / precursor cells or progenitor cells are used to differentiate in vivo to more advanced evolutionary and / or mature endocrine cells. Such agents include, but are not limited to, a retinoid or retinoic acid, BMP, Nicotinamide, IGF, Hedgehog proteins and the like. In a preferred embodiment, TTNPB (4 - [(E) -2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1-propenyl] benzoic acid or Arotinoid) is a selective and highly potent retinoic acid analog with affinity for retinoic acid receptors (RAR) a, pyy, which are nuclear transcription factors. TTNPB and other retinoic acid and retinoic acid analogs produce ligand-activated transcription of genes that possess retinoic acid sensitive elements. Accordingly, other agents or ligands capable of activating retinoic acid sensitive elements or binding to any of the RARs are potentially useful for and adaptable to the present invention to enhance the differentiation of endocrine cells.

In another embodiment, cell cultures of phase 5 or phase 6 or phase 7 can also be treated with Matrigel alone or in combination with a rho-kinase inhibitor to improve cell adhesion. Alternatively, a rho-kinase inhibitor can be added in phases 5, 6 or 7 at concentrations sufficient to enhance cell survival, cell mass and performance. Alternatively, cell cultures can be dissociated and re-associated during or between phases 6 and 7 to eliminate or deplete unwanted cell types that include, but are not limited to, the subpopulation of non-endocrine multipotent pancreatic progenitors (CHGA-). See example 14.

In yet another embodiment, any of phases 1-7 can be extended 1,2, 3, 4, 5, 6, 7, 8, 9, 10 more days or shortened by 1, 2, 3, 4, 5, 6, 7 or more days For example, in a preferred embodiment and according to Table 17, phase 6 occurs for about 6 days and phase 7 for about 9 days. However, both phases can be shortened to accommodate the production of an endocrine parent / precursor, for example, to lengthen and prolong to 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more days in order to produce an endocrine cell with more advanced development.

In another embodiment, methods are provided for producing a more advanced PEC in development, or pancreatic endocrine progenitor / precursor cell or pancreatic endocrine. In one aspect of the invention, an endocrine cell is provided that expresses only a pancreatic hormone that is positive in both PDX1 and NKX6.1, for example, an immature beta cell that jointly expresses INS, PDX1 and NKX6.1. In another aspect, an evolutionarily advanced cell may be a PEC culture that consists primarily of non-endocrine pancreatic progenitor cells that express at least PDX and NKX6.1, but that does not consist or that minimally consists of cells compromised with the endocrine lineage (CHGA + ) that express at least CHGA +, NGN3 and / or NKX2.2. These PEC cultures are evolutionarily advanced, because they are considered more similar to those observed in vivo in the development studies of pancreatic differentiation. See Jorgensen et al. (2007), "An Illustrated Review of Early Pancreas Development in the Mouse", Endocrine Reviews 28 (6): 685-705, and Rukstalis and Habener (2009), "Neurogenin3: A master regulator of pancreatic islet differentiation and regeneration", Islets 1 (3): 177-184. Another example of a cell or an evolutionarily advanced cell culture is one that has been dissociated and reagregated or re-associated so that the reacgregated cell culture consists of a more homogeneous population of cells. This population of more homogeneous cells, in one aspect of the invention, does not comprise cell types in earlier phases, for example, pancreatic or endocrine endocrine progenitor / precursor cells that mainly comprise cells expressing a single hormone that are both PDX1 positive as in NKX6.1, but which do not include cells or subpopulations in phases 3 and 4 or subpopulations of PEC or PEC, which include, but are not limited to, non-endocrine multipotent pancreatic progenitor cells (CHGA-), or triple negative / residual cells (CHGA- / PDX1- / NKX6.1-) and the like, or cells that express multiple hormones (i.e., cells that express more than one hormone in any type of cell).

In another embodiment, the cell cultures provided herein are preferably properly specified, which has a specific and true meaning in the art of evolutionary biology. In the context of evolutionary biology, it is the mechanism by which cells obtain different destinations or are specified or specified correctly. Since differentiation of in vitro cell culture lacks temporal organization and typical signals from in vivo evolutionary studies , the use and dependence of cell markers (surface or internal markers such as transcription factors), in particular, markers Distinctive or outgoing cell indicative of a specific cell type, are essential. Therefore, the reference to duly specified cells, cell cultures, subpopulations and populations means that these cells have different destinations, and that those destinations are safer and are determined based on the expression of their marker, the profile of the markers that they express and, equally important, the markers they do not express. The proper specification of in vitro cell cultures depends on in vivo evolutionary studies of similar cells, therefore, in a preferred embodiment, in vivo evolutionary studies are a guide for in vitro differentiation and characterization of suitably specified cells.

In some embodiments of the invention described herein, exendin 4 is provided to the cell culture or cell differentiation population at approximately the same time as the gamma secretase inhibitor. In certain embodiments, exendin 4 is provided to be present in the cell culture or in the cell population at a concentration of at least about 0.1 ng / ml to 1,000 ng / ml.

It will be appreciated that the expression of the NGN3, NKX2.2 and / or PAX4 markers is induced in a range of different levels in endocrine progenitor / precursor cells depending on differentiation conditions. As such, in some embodiments described herein, the expression of the NGN3, NKX2.2 and / or PAX4 marker in cells or in endocrine progenitor / precursor cell populations is at least about 2 times greater than at least about 10,000 times. superior to the expression of the NGN3, NKX2.2 and / or PAX4 marker in non-endocrine progenitor / precursor cell cells or populations, for example, pluripotent stem cells, definitive endodermal cells, upper digestive tract endodermal cells positive in PDX1, immature cells expressing pancreatic islet hormone, mature cells expressing pancreatic islet hormones, extraembryonic endodermal cells, mesodermal cells and / or ectodermal cells. In other embodiments, the expression of the NGN3, NKX2.2 and / or PAX4 marker in cells or in endocrine progenitor / precursor cell populations is at least about 4 times higher, at least about 6 times higher than 10,000 times higher than the expression of the NGN3, NKX2.2 and / or PAX4 marker in non-endocrine progenitor / precursor cell cells or populations, for example, pluripotent stem cells, definitive endodermal cells, upper digestive tract endodermal cells positive in PDX1, immature cells expressing hormone of the Pancreatic islet, mature cells that express hormones from the pancreatic islet, extra-embryonic endodermal cells, mesodermal cells and / or ectodermal cells. In some embodiments, the expression of the NGN3, NKX2.2 and / or PAX4 marker in cells or in endocrine progenitor / precursor cell populations is infinitely superior to the expression of the NGN3, NKX2.2 and / or PAX4 marker in cells or populations of non-endocrine progenitor / precursor cells, for example, pluripotent cells such as iPS cells and hES cells, definitive endodermal cells, positive upper digestive tract endodermal cells in PDX1, immature cells expressing pancreatic islet hormone, mature cells expressing hormones of the pancreatic islet , extraembryonic endodermal cells, mesodermal cells and / or ectodermal cells.

Other embodiments of the present invention relate to compositions, such as cell cultures or cell populations, comprising human cells, including human endocrine progenitor / precursor cells, in which the expression of the NGN3 marker is superior to the expression of the AFP marker, SOX7. , SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6 in at least about 2% of human cells. In other embodiments, the expression of the NGN3 marker is superior to the expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6 marker by at least about 5% to 98% of human cells. In some embodiments, the percentage of human cells in cultures or cell populations, in which the expression of NGN3 is greater than the expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, marker, GCG, SST, GHRL and / or Pa X6, is calculated without taking into account the feeder cells.

It will be appreciated that some embodiments of the present invention relate to compositions, such as cell cultures or cell populations, comprising human endocrine progenitor / precursor cells, in which the expression of NKX2.2 and / or PAX4 is greater than the expression of AFP marker, SOX7, SOX1, ZIC1, NFM, Ma Fa, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6 in at least about 2% to more than at least about 98% of human cells. In some embodiments, the expression of NKX2.2 and / or PAX4 is superior to the expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL and / or pAX6 marker at al less than about 5% of human cells to 98% of human cells. In some embodiments, the percentage of human cells in cultures or cell populations, in which the expression of NKX2.2 and / or PAX4 is greater than the expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA marker, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6, is calculated without taking into account the feeder cells.

Using the processes described herein, compositions comprising endocrine progenitor / precursor cells substantially free of other cell types can be produced. In some embodiments of the present invention, cell populations or cultures of endocrine progenitor / precursor cells produced by the methods described herein are substantially free of cells that significantly express AFP, SOX7, SOX1, ZIC1 and / or markers. NFM In some embodiments, the endocrine progenitor / precursor cell populations of cell cultures produced by the methods described herein are substantially free of cells that significantly express the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA markers. INS, GCG, SST, GHRL and / or PAX6.

In yet other processes for the production of immature cells expressing pancreatic islet hormones disclosed herein, HGF is provided to the cells so that it is present in the cell culture or in the cell population at concentrations sufficient to enhance the differentiation of at least part of the endocrine progenitor / precursor cells to immature cells that express hormones from the pancreatic islet. In some embodiments, HGF is present in the cell culture or in the cell population at a concentration of at least about 1 ng / ml, of at least about 5 ng / ml to 1,000 ng / ml.

In yet other processes for the production of immature cells of pancreatic islet hormones described herein, IGF1 is provided to the cells so that it is present in the cell culture or in the cell population at concentrations sufficient to enhance the differentiation of less a part of the endocrine progenitor / precursor cells to immature cells expressing pancreatic islet hormones. In some embodiments, IGF1 is present in the cell culture or in the cell population at a concentration of at least about 1 ng / ml to 1,000 ng / ml.

In certain embodiments of the processes for producing immature cells expressing pancreatic islet hormones as described herein, one or more of nicotinamide, exendin 4, HGF and IGF1 are provided after one or more differentiation factors previously provided. have been removed from cell cultures. In other embodiments, one or more nicotinamide, exendin 4, HGF and IGF1 are provided to a cell culture or to a cell population comprising one or more differentiation factors that were previously provided or provided at almost the same time as one or more of nicotinamide. , exendina 4, HGF and IGF1. In preferred embodiments, the differentiation factors that were provided above or that were provided at approximately the same time as one or more of nicotinamide, exendin 4, HGF and IGF1 include, but are not limited to, DAPT, FGF-10, KAAD-cyclopamine, Activin A, Activina B, BMP4 and / or RA.

Other embodiments of the present invention relate to compositions, such as cell cultures or cell populations, comprising human cells, including immature human cells expressing hormones of the pancreatic islet, in which the expression of the MAFB, SYP, CHGA, NKX2 marker. 2, ISL1, Pa X6, NEUROD, PDX1, HB9, GHRL, IAPP, INS GCG, SST, PP and / or C-peptide is superior to the expression of the NGN3, MAFA, MOX1, CER, POU5F1, AFP, SOX7, SOX1 marker , ZIc 1 and / or Nf M in at least about 2% of human cells. In other embodiments, the expression of the MAFB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, PDX1, HB9, GHRL, IAPP INS GCG, SST, PP and / or C-peptide marker is superior to the expression of the NGN3 marker , MAFA, MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and / or NFM in at least about 5% of human cells, in at least about 10% of human cells to 95% of human cells or in at least about 98% of human cells.

In certain embodiments of the present invention, cell cultures and / or cell populations comprising immature cells expressing pancreatic islet hormones also include a medium comprising one or more secreted hormones selected from ghrelin, insulin, somatostatin and / or glucagon. In other embodiments, the medium comprises C-peptide. In a preferred embodiment, the concentration of one or more hormones or C-peptide secreted in the medium ranges from at least about 1 pmol of ghrelin, insulin, somatostatin, glucagon or C / pg peptide of Cellular DNA at at least about 1,000 picomoles (pmol) of ghrelin, insulin, somatostatin, glucagon or C / pg peptide of cellular DNA.

In vivo insulin production method

In some embodiments, in vitro derived pancreatic progenitors or pancreatic endodermal type cells PDX-1 or equivalent thereof described above are transplanted into a mammalian subject. These methods are described in detail in the international application PCT / US2007 / 015536, entitled "METHODS OF PRODUCING PANCREATIC HORMONES", and Kroon et al. (2008) supra. In a preferred embodiment, the mammalian subject is a human subject. Particularly preferred subjects are those who have been identified as having a condition that limits the subject's ability to produce sufficient levels of insulin in response to physiologically high concentrations of blood glucose. Those skilled in the art can easily determine a range of blood glucose levels that constitutes a physiologically high level of blood glucose for any particular mammalian species. Any persistent level of blood glucose that produces a recognized disease or condition is considered a physiologically high level of blood glucose.

Additional embodiments of the present invention relate to an in vivo insulin secretory cell that is derived from an in vitro pluripotent stem cell or its progeny, for example, multipotent cells, such as upper digestive tract endodermal cells positive in PDX-1 , pancreatic or pancreatic endodermal progenitor cell in PDX-1, an endocrine progenitor / precursor cell, such as a positive endocrine progenitor / precursor cell in NGN3 or a functional differentiated hormone secreting cell, such as an insulin, glucagon, somatostatin, ghrelin or pancreatic polypeptide secretory cell. Any of the multipotent or terminal differentiated cells described above can be transplanted to the host or mammal, and mature into physiologically functional hormone secreting cells, such as insulin secreting cells, in response to host blood glucose levels. In preferred embodiments, the cell does not form a teratoma in vivo, and if so formed, it remains located in the area of the transplant and can be easily removed or removed. In especially preferred embodiments, the cell does not contain any karyotypic abnormalities during the in vitro differentiation process , or when the mammal is transplanted in vivo, or when mature and developed on functional islets in vivo.

In addition, although the embodiments described herein refer to a genetically modified or genetically engineered pluripotent cell, multipotent or differentiated cell derived from the pluripotent cell, such as a human iPS cell, based on the description provided herein , it is anticipated that, since iPS cells show similar physiology and gene marker expression profiles compared to hES cells and hES derived cells, they will have similar physiological characteristics in vivo.

Pancreatic progenitor encapsulation method

In some embodiments, the pluripotent, multipotent and differentiated cell composition described herein. The document can be encapsulated in a biological and / or non-biological mechanical device, in which the encapsulated device separates and / or isolates the cellular compositions from the host.

The encapsulation methods are described in detail in the US application. 61 / 114,857, filed on Friday, November 14, 2008, entitled "ENCAPSULATION OF PANCREATIC PROGENITORS DERIVED FROM HES CELLS", and the US application. n. 61 / 121.086, filed on December 12, 2008, entitled "ENCAPSULATION OF PANCREATIC ENDODERM CELLS".

In one embodiment, the encapsulation device contains the pluripotent derived cells, for example, positive upper digestive tract endodermal cell in PDX-1, positive pancreatic progenitor or endodermal cell in PDX-1, an endocrine or endocrine progenitor / precursor cell, such as a positive endocrine progenitor / precursor cell in NGN3 or a functional differentiated hormone secreting cell, such as an insulin, glucagon, somatostatin, ghrelin or pancreatic polypeptide secreting cell, in a semipermeable membrane that prevents the passage of the population of transplanted cells , retaining them in the device, while at the same time allowing the passage of certain secreted polypeptides, for example, insulin, glucagon, somatostatin, ghrelin, pancreatic polypeptide and the like. Alternatively, the device has a plurality of membranes, including a vascularizing membrane.

Use of agents to enhance and promote growth, survival, proliferation and cell-cell adhesion of human pluripotent stem cells, for example, hES cells and iPS cells

Cellular regulation can be affected through the transduction of extracellular signals through the membrane, which, in turn, modulates the biochemical pathways within the cell. Protein phosphorylation represents a course whereby intracellular signals propagate from one molecule to another, which ultimately produces a cellular response. These signal transduction cascades are highly regulated and often overlap, as evidenced by the existence of many protein kinases and phosphatases. It has been reported that, in humans, tyrosine kinase proteins are known to have an important role in the development of many disease states, including diabetes, cancer, and have also been linked to a wide variety of congenital syndromes. Serine / threonine kinases, for example, Rho kinases, are a class of enzymes, which, if inhibited, may be relevant for the treatment of human diseases, including diabetes, cancer, and various inflammatory cardiovascular disorders and AIDS. Most inhibitors identified to date act at the ATP binding site. Such inhibitors competitive with ATP have demonstrated selectivity by virtue of their ability to target the most poorly conserved areas of the ATP binding site.

The Rho kinase family of small GTP-binding proteins contains at least 10 members, including Rho A-E and G, Rac 1 and 2, Cdc42 and TC10. Inhibitors are often referred to as ROK or ROCK inhibitors, or Rho-kinase inhibitors, and are used interchangeably herein. The effector domains of RhoA, RhoB and RhoC have the same amino acid sequence and appear to have similar intracellular targets. Rho kinase functions as a Rho primary chain downstream mediator and exists as two isoforms: a (ROCK2) and p (ROCK1). Rho kinase family proteins have a catalytic domain (kinase) in their N-terminal domain, a supercoiled domain in their middle and a putative pleckstrin (PH) homologous domain in their C-terminal region. The Rho-binding domain of ROCK is located in the C-terminal portion of the supercoiled domain, and the binding of Rho's GTP-bound form causes an increase in kinase activity. The Rho / Rho-kinase-mediated pathway plays an important role in signal transduction initiated by many agonists, including angiotensin II, serotonin, thrombin, endothelin-1, norepinephrine, platelet-derived growth factor, ATP / ADP and extracellular nucleotides , and urotensin II. Through the modulation of its target effectors / substrates, Rho kinase plays an important role in various cellular functions, including smooth muscle contraction, the organization of the actin cytoskeleton, cell adhesion, and gene motility and expression. Due to the role that Rho kinase proteins play in mediating a series of cellular functions that are considered to be associated with the pathogenesis of arteriosclerosis, these kinase inhibitors may also be useful for the treatment or prevention of various arteriosclerotic cardiovascular diseases. and that participate in endothelial contraction.

In some embodiments, agents that enhance and / or support cell growth, survival, proliferation and cell-cell adhesion are added to various conditions of cell culture media, including, but not limited to, Rho-kinase inhibitors. Y-27632, Fasudil (also known as HA1077), H-1152P and ITS (insulin / transferrin / selenium; Gibco), Wf-536, Y-30141 (described in US Patent No. 5,478,838 ) and its derivatives, and non-ROCK-encoding nucleic acid, nucleic acid that induces RNA interference (eg, siRNA), competitive peptides, antagonistic peptides, inhibitory antibodies, ScFV antibody fragments, dominant negative variants and expression vectors of the same. In addition, since there are other low molecular weight compounds that are known as ROCK inhibitors, said compounds or derivatives thereof can also be used in the present invention (for example, see US patent applications n. 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344 and 20030087919, and international patent publications No. 2003/062227, 2003./059913, 2003/062225, 2002/076976 and 2004/039796).

In the present invention, a combination of one or two or more of the ROCK inhibitors can also be used.

These agents function, in part, by enhancing the re-association of cultures of dissociated hES cells, of iPS or of differentiated cells, for example, definitive endodermal, upper digestive tract, pancreatic endodermal, pancreatic epithelium, pancreatic progenitor populations, endocrine progenitor populations and the like Also, agents can function when cell dissociation is not performed. Increased growth, survival, proliferation and cell-cell adhesion of human pluripotent stem cells were achieved regardless of whether the cells were produced from suspended cell aggregates or adherent plate cultures (with or no extracellular matrix components, with or without serum, with or without fibroblast feeders, with or without FGF, with or without Activin). Increasing the survival of these cell populations facilitates and improves the purification systems using a cell sorter and, therefore, allows better cell recovery. The use of Rho kinase inhibitors such as Y27632 may allow the expansion of differentiated cell types by enhancing their survival during serial pass-through of dissociated or cryogenic preservation cells. Although Rho kinase inhibitors such as Y27632 have been tested in human pluripotent stem cells (for example, hES and iPS cells) and differentiated cells thereof, Rho kinase inhibitors can be applied to other cell types, by For example, in general, epithelial cells that include, but are not limited to intestinal, pulmonary, thymus, kidney and neuronal cell types such as pigmented retinal epithelium.

The concentration of the ROCK inhibitor in the cell culture medium is not limited to what is described in the following examples, provided that the concentration can achieve the desired effects, such as enhancement, increase and / or growth promotion, of survival, proliferation and cell-cell adhesion of cells. One skilled in the art will recognize that optimization of various ROCK inhibitors may be necessary under various conditions. For example, when Y-27632 is used, a preferable concentration may vary between about 0.01 and about 1,000 pM, more preferably from about 0.1 to about 100 pM, and even more preferably from about 1.0 to about 50 pM , and most preferably from about 5 to 20 pM. When Fasudil / HA1077 is used, it can be used at about two or three times the concentration of Y-27632 mentioned above. When H-1152 is used, it can be used in about a fraction, for example, Approximately 1/10, 1/20, 1/30, 1/40, 1/50 or 1/60 of the amount of the aforementioned concentration of Y-27632. The concentration of ROCK inhibitor used will depend, in part, on the bioactivity and potency of the inhibitor, and on the conditions in which it is used.

The time and period of treatment with the ROCK inhibitor may or may not be limited depending on the desired effects, such as enhancement, enhancement and / or promotion of growth, survival, proliferation (cell mass) and adhesion. cell-cell However, the addition of a ROCK inhibitor can also affect differentiation in surprising ways, as best described in Example 7. The following examples describe cultures of human pluripotent stem cells and / or cultures of differentiated cells treated for approximately 12 hours, 24 hours, 48 hours or more.

The density of human pluripotent stem cell cultures treated with the ROCK inhibitor is also not limited to the extent that it is a density at which desired effects are achieved, such as enhancement, enhancement and / or growth promotion. , survival, proliferation and cell-cell adhesion of cells. The cell density of the seeded cells can be adjusted depending on several factors, including, but not limited to, the use of adherent or suspended cultures, the specific recipe of the cell culture medium used, the growth conditions and the contemplated use of the cells. cultivated Examples of cell culture densities include, but are not limited to, 0.01 x 105 cells / ml, 0.05 x 105 cells / ml, 0.1 x 105 cells / ml, 0.5 x 105 cells / ml, 1 , 0 x 105 cells / ml, 1.2 x 105 cells / ml, 1.4 x 105 cells / ml, 1.6 x 105 cells / ml, 1.8 x 105 cells / ml, 2.0 x 105 cells / ml, 3.0 x 105 cells / ml, 4.0 x 105 cells / ml, 5.0 x 105 cells / ml, 6.0 x 105 cells / ml, 7.0 x 105 cells / ml, 8, 0 x 105 cells / ml, 9.0 x 105 cells or 10.0 x 105 cells / ml or more, for example, up to 5 x 107 cells / ml or more, or any intermediate value, have been grown with good growth, cell-cell survival, proliferation and adhesion of cells.

Use of agents that activate members of the TGFB receptor family

In yet another embodiment, agents that activate the member of the TGFp receptor family include members of the TGFp growth factor superfamily, are described herein. As used herein, "TGFp superfamily member" or equivalent thereof refers to more than 30 structurally related proteins, including subfamilies that include TGFp1, TGFp2, TF-p3, GDF-15, GDF- 9, BMP-15, BMP-16, BMP-3, GDF-10, BMP-9, BMP-10, GDF-6, GDF-5, GDF-7, BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, BMP-4, GDF-3, GDF-1, GDF 11, GDF8, Activinas pC, pE, pA and pB, BMP-14, GDF-14, MIS, Alpha inhibit, Lefty1, Lefty2 , GDNF, Neurteurin, Persefin and Artemina. See Chang et al. (2002) Endocnne Rev. 23 (6): 787-823.

A member of the TGFp family can be replaced by, or used in combination with, a TGFp signaling pathway activator, which is a compound that stimulates one or more of the polypeptides or interactions that participate in transduction or performance. of other changes in the properties of a cell in response to a member of the TGFp family. A TGFp signaling pathway includes the members of the TGFp family themselves. Members of the TGFp superfamily transmit signals to the nucleus by signaling through serine-threonine kinase type II and I receptors and intracellular effectors known as Smads. These receivers they are divided into two subfamilies known as type I and type II receptors that act cooperatively to bind the ligand and transduce the signal (Attisano et al., Mol Cell Biol 16 (3), 1066-1073 (1996)). The ligands bind to type I and II receptors on the cell surface, enhancing the activation of type I receptor through phosphorylation. This activated complex, in turn, activates intracellular Smads, which assemble complexes of multiple subunits that regulate transcription. Members of the TGFbeta superfamily are divided into two signaling subgroups: those functionally related to TGFp / Activina and those related to the BMP / GDF subfamily. It is thought that most TGFp ligands bind first to a type II receptor, and this type II ligand / receptor complex then recruits or phosphorylates a type I receptor (Mathews, LS, Endocr Rev 15: 310- 325 (1994); Massague, Nature Rev: Mol Cell Biol. 1, 169-178 (2000)). Type II receptor kinase by phosphorylation and activation of type I receptor kinase, which then phosphorylates and activates Smad proteins. TGFp / Activin ligands bind to TGFp and Activin type II receptors and can activate Smad-2 and -3. Nodal and Lefty signal through this Activina type pathway. BMP / GDF ligands bind to type II BMP receptors and can activate Smads 1.5 and 8. See Derynck, R et al. Cell 95, 737-740 (1998)). After ligand stimulation, Smads proteins move to the nucleus and function as components of transcription complexes.

TGFp signaling is regulated positively and negatively through various mechanisms. Positive regulation amplifies the signals to a level sufficient for biological activity. TGFp superfamily ligands bind to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates receptor-regulated SMADs (R-SMAD, for example, SMAD1, SMAd 2 , SMAD3, SMAD5 and SMAD8) which can then link the common mediator Smad or co-SMAD. The R-SMAD / coSMAD complexes accumulate in the nucleus, in which they act as transcription factors and participate in the regulation of target gene expression. For example, growth differentiation factors include 1, 2, 3, 5, 6, 7, 8, 9, 10, 11 and 15. And, in a preferred embodiment, GDF8 and GDF11, are members of the TGFp family which are also activators of the TGFp signaling pathway (positive regulation) and act by binding to the part of the binding domain to the extracellular ligand of the ActRII receptor and then forming a complex with ActRI, which leads to inhibition of the negative regulator of Smad7 and the phosphorylation of the Smad2 / Smad3 complex. The Smad2 / Smad3 complex is associated with Smad4 to regulate the expression of certain genes.

As with the use of any agent, the concentration of any member of the TGFp superfamily in the cell culture medium is not limited to that described in the following examples, provided that the concentration can achieve the desired effects to activate a member from the family of TGFp receptors, for example. For example, when Activinas, for example, Activina A and / or B, or GDF8 and GDF-11 are employed, a preferable concentration may vary from about 10 to about 300 nM, more preferably from about 50 to about 200 nM, and even more preferably from about 75 to about 150 nM, and most preferably, from about 100 to 125 nM. A person skilled in the art can easily test any concentration and the use of conventional techniques in the field can determine the efficacy of said concentration, for example, by evaluating the differentiation by determining the expression and non-expression of a panel of gene markers for Any type of cell.

Use of agents to produce endocrine cells

In one embodiment, the present invention provides methods for creating populations and / or subpopulations of pancreatic endodermal lineage type, specifically PEC and / or pancreatic endocrine lineage cells, using a Notch pathway inhibitor, including, but not limited to, an inhibitor of gamma secretase, in an amount effective to enhance endocrine differentiation and / or induce the expression of certain markers that are distinctive of endocrine cells and are indicative of endocrine differentiation. Numerous gamma secretase inhibitors have been described. See, for example, US Pat. No. 6,756,511; 6,890,956; 6,984,626; 7,049,296; 7,101,895; 7,138,400; 7,144,910; and 7,183,303. Gamma secretase inhibitors can be easily obtained, for example, from Calbiochem (La Jolla, California): gamma secretase I inhibitor, (GSI I), Z-Leu-Leu-Norleucine-CHO; gammasecretase II inhibitor; gamma secretase III inhibitor, (GSI III), N-Benzyloxycarbonyl-Leu-leucinal; gamma secretase IV inhibitor, (GSI IV), N- (2-Naphthoyl) -Val-phenylalaninal; gamma secretase V inhibitor, (GSI V), N-benzyloxycarbonyl-Leu-phenylalaninal; gamma secretase VI inhibitor, (GSI VI), 1- (S) -endo-N- (1,3,3) -Trimethylbicyclo [2.2.1] hept-2-yl) -4-fluorophenyl sulfonamide; gamma secretase VII inhibitor, (GSI VII), Methyloxycarbonyl-LL-CHO; gamma secretase IX inhibitor, (GSI IX), (DAPT), N- [N- (3,5-difluoropheacetyl-L-alanyl)] - S-phenylglycine t-butyl ester; gamma secretase X inhibitor, (GSI X), {1 S-benzyl-4R- [1- (1 S-carbamoyl-2-phenethylcarbamoyl) -1 S-3-methylbutylcarbamoyl] -2R- tert-butyl ester hydroxy-5-phenylpentyl} carbamic; gamma secretase XI inhibitor, (GSI XI), 7-amino-4-chloro-3-methoxyisocumarine; gamma secretase XII inhibitor, (GSI XII), Z-Ile-Leu-CHO; gamma secretase XIII inhibitor, (GSI XIII), Z-Tyr-Ile-Leu-CHO; gamma secretase XIV inhibitor, (GSI XIV), Z-Cys (t-Bu) -Ile-Leu-CHO; gamma secretase XVI inhibitor, (GSI XVI), N- [N-3,5-difluoropheacetyl] -L-alanyl-S-phenylglycine methyl ester; gamma secretase XVII inhibitor, (GSI XVII), WPE-III-31C); gamma secretase XIX inhibitor, (GSI XIX), (2S, 3R) -3- (3,4-Difluorophenyl) -2- (4-fluorophenyl) -4-hydroxy-N - ((3S) -2-oxo -5-phenyl-2,3-dihydro-1H-benzo [e] [1,4] diazepin-3-yl) -butyramide; gamma secretase XX inhibitor, (GSI XX), (Dibenzazepine (DBZ)), (S, S) -2- [2- (3,5-Difluorophenyl) acetylamino] -N- (5-methyl-6-oxo- 6,7-dihydro-5H-dibenzo [6, d] azepin-7-yl) propionamide, gamma secretase XXI inhibitor, (GSI XXI), (S, S) -2- [2- (3.5 -Difluorophenyl) -acetylamino] -N- (1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo [e] [1,4] diazepin-3-yl) -propionamide; gamma40 secretase I, N-trans-3,5-Dimethoxycinnamoyl-Ileleucinal inhibitor; gamma40 secretase II inhibitor, N-tert-butyloxycarbonyl-Gly-Val-Valinal; and isovaleryl-VV-Sta-A-Sta-OC ³ .

In one aspect, the gamma secretase inhibitor is selected from the group consisting of one or more of: Z-Leu-Leu-Norleucine-CHO, N-Benzyloxycarbonyl-Leu-leucinal, N- (2-Naphthoyl) -Val- phenylalaninal, N-Benzyloxycarbonyl-Leufenilalaninal, 1- (S) -endo-N- (1,3,3) Trimethylbicyclo [2.2.1] hept-2-yl) -4-fluorophenyl-sulfonamide, Methyloxycarbonyl-LL-CHO, N- [N- (3,5-difluoropheacetyl-L-alanyl)] - S-phenylglycine f-butyl ester {1S-benzyl-4R- [1- (1S-carbamoyl-2-) phenethylcarbamoyl) -1S-3-methylbutylcarbamoyl] -2R-hydroxy-5-phenylpentyl} carbamic, 7-amino-4-chloro-3-methoxyisocumarine, Z-lle-Leu-CHO, Z-Tyr-lle-Leu-cHo, Z-Cys (t-Bu) -lle-Leu-CHO, N- [N-3,5-difluoropheacetyl] -L-alanyl-S-phenylglycine methyl ester, (2S, 3R) -3- (3,4 -Difluorophenyl) -2- (4-fluorophenyl) -4-hydroxy-N - ((3S) -2-oxo-5-phenyl-2,3-dihydro-1H-benzo [e] [1,4] diazepin- 3-yl) -butyramide, (S, S) -2- [2- (3,5-Difluorophenyl) -acetylamino] -N- (1-methyl-2-oxo-5-phenyl-2-, 3-dihydro -1H-benzo [e] [1,4] diazepin-3-yl) -propionamide, N-fra ns-3,5-Dimethoxycinnamoyl-Ile-leucinal, N-fercbutyloxycarbonyl-Gly-Val-Valinal, Isovaleril-VV-Sta-A-Sta-OCH ³ , and in which the renal disease is a glomerular kidney disease or a disease tubular renal In another aspect of the present invention, about 0.5 to 10 mM, 0.5 to 5 mM, 0.5 to 3 mM, preferably 0.5 to 1 mM gamma secretase inhibitor is used in any or all phases 1-7 described herein and in accordance with Table 8 and 17.

In one embodiment, insulin growth factor (IGF) is part of a complex system that cells use to communicate with their physiological environment and, therefore, can be used to differentiate PSC cells to PEC and / or lineage cells. endocrine pancreatic This complex system (often called the "axis" of IGF) consists of two cell surface receptors (IGF1R and IGF2R), two ligands (insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2)), a family of six high affinity IGF-binding proteins (IGFBP-1 to IGFBP-6), as well as associated IGFBP degradation enzymes, collectively referred to as proteases. It is known that IGFs bind to IGF1R, the insulin receptor, the IGF-2 receptor, the insulin-related receptor and possibly other receptors. Therefore, in one aspect of the present invention, growth factors that bind to these receptors include, but are not limited to, IGF1 and iGF2, since any growth factor, agent or morphogen that binds to either or a combination of these receptors has potentially the same physiological effect as described and anticipated herein. In another aspect of the present invention, about 5 to 200 ng / ml, 10 to 100ng / ml, 10 to 75 ng / ml, preferably 10 to 50 ng / ml, preferably 20 to 50 ng / ml are used of IGF in any or all phases 1-7 described herein, and in accordance with Table 8 and 17.

In one embodiment, the platelet-derived growth factor (PDGF) is used to differentiate PSC cells to PEC and / or cells committed to the endocrine lineage (CHGA +). PDGF is an important protein growth factor that has been widely described as a potent mitogen of numerous cell types. PDGF belongs to a family of dimeric isoforms of polypeptide chains, A, B, C and D that act through different tyrosine kinase receptors: PDGFR-a and PDGFR-p. In one aspect of the invention, the ligand is a PDGF or functional equivalent thereof that includes, but is not limited to PDGFaa, PDGFab or PDGFbb, which bind to the two types of receptors. In another aspect of the present invention, about 5 to 100 ng / ml, 10 to 75 ng / ml, 10 to 50 ng / ml, preferably 10 to 20 ng / ml PDGF are used in any or all phases 1-7 described herein, and in accordance with Table 8 and 17.

In one embodiment, Nicotinamide, also known as niacinamide and nicotinic acid amide, is the nicotinic acid amide (vitamin B3 / niacin), is used to differentiate PSC cells to PEC and / or pancreatic endocrine lineage cells. Nicotinic acid, also known as niacin, is converted to nicotinamide in vivo, and, although niacin and nicotinamide are identical in their vitamin functions, nicotinamide does not have the same pharmacological and toxic effects of niacin. Accordingly, other derivatives of vitamin B or vitamin B that function similarly to nicotinamide are made in the present invention. In another aspect of the present invention, about 5 to 100 ng / ml, 10 to 75 ng / ml, 10 to 50 ng / ml, preferably 10 to 20 ng / ml nicotinamide are used in any or all of the phases 1-7 described in this document and in accordance with tables 8 and 17.

In another embodiment, the Hedgehog family of proteins is used to differentiate PSC cells to PEC and / or pancreatic endocrine lineage cells. The hedgehog family of vertebrate intercellular signaling molecules provided by the present invention consists of at least four members, including, but not limited to, Desert hedgehog (Dhh), Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Moonrat hedgehog (Mhh ). In one aspect, about 5 to 200 ng / ml, 10 to 200 ng / ml, 10 to 150 ng / ml, preferably 10 to 100 ng / ml of a hedgehog protein are used in any or all phases 1- 7 described in this document and in accordance with Table 8 and 17.

Having described the present invention in general, it can be better understood by referring to certain specific examples provided herein for purposes of illustration only, and which are not intended to be limiting.

Examples

It should also be understood that the foregoing refers to preferred embodiments of the present invention, and that numerous changes can be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which should not be construed as limitations in any way. within reach of it. On the contrary, it should also be understood that various other embodiments, modifications and equivalents thereof may be used, which, after reading the description contained herein, can be suggested to those skilled in the art.

Example 1

DIFFERENTIATION OF HUMAN iPS CELLS TO PANCREATIC PROGENITATING CELLS AND ENDOCRINE CELLS THROUGH DEFINITIVE AND INTERMEDIATE ENDODERMIC PRODUCTS

Human-induced pluripotent stem cells (iPS) were differentiated into suspension aggregates using a four (4) phase procedure over approximately 2 weeks (or 14 days) to generate a population of pancreatic cell types, including cells pancreatic progenitors, endocrine progenitor cells and endocrine cells that express hormones. The strains of human iPS cells employed herein were provided by S. Yamanaka, Kyoto University, Japan and Cellular Dynamics International, Inc. (CDI).

The iPS cells described herein were provided first by Shinya Yamanaka and then by the CDI. Indifferentiated iPS cells were cultured in mitotically inactivated mouse embryo fibroblasts or, preferably, feeder-free (without fibroblast feeder cell layer) in DMEM / F12 containing 20% deactivated serum replacement. Differentiation was initiated by dissociating undifferentiated iPS cells into individual cells using Accutase. Cell samples were taken for RNA isolation and analysis. The cells were resuspended at 1-2 million cells per milliliter in RPMI + 0.2% vol / vol FBS containing a 1: 5,000 dilution of insulin-transferrin-selenium (ITS), Activin A (100 ng / ml), wnt3a (50 ng / ml) and rho-kinase or ROCK inhibitor, Y-27632, at 10 pM, were placed in a 6-well plate of ultra low fixation, placed on a rotating platform and centrifuged at about 100 rpm. The cultures were centrifuged at 100 rpm during the rest of the differentiation process with the daily exchange of the medium. The growth, passage and proliferation of iPSC is essentially as described in US Pat. UU. No. 7,961,402 and 8,211,699.

The methods described herein to produce suspension cultures of pluripotent cell aggregates, for example, hES or iPS cells, and cells derived from pluripotent cells, are essentially described in PCT / US2007 / 062755, filed February 23 of 2007 and entitled "Compositions and methods for culturing differential cells", and document PCT / US2008 / 080516, filed on October 20, 2008, and entitled "Methods and compositions for feeder-free pluripotent stem cell media containing human serum".

The methods described herein can be facilitated by first coating the culture vessels with an extracellular matrix, for example, as described in US Pat. No. 6,800,480 to Bodnar et al. and assigned to Geron Corporation. The methods, like other methods for culturing other pluripotent stem cells, for example, hES and iPS cells, can be performed using soluble human serum as essentially described in the US application. UU. PCT / US2008 / 080516, filed on October 20, 2008, and entitled "Methods and compositions for feederfree pluripotent stem cell media containing human serum".

The methods described herein can be facilitated by exogenously adding fibroblast growth factor (FGF) supplied from a source other than just a fibroblast feeder layer as described in US Pat. 7,005,252 to Thomson, J. and assigned to the Wisconsin Alumni Research Foundation (WARF).

During approximately the first 24 hours of rotation, the individual cells adhered together forming cell aggregates, and sufficient cell samples were taken for RNA isolation and analysis. Cellular aggregates varied between approximately 60 micrometers and 120 micrometers in diameter. Approximately 1 day (or 24 hours) after the iPS cell samples were placed on the rotation platform, the cultures were fed with RPMI + 0.2% vol / vol FBS containing a 1: 5000 dilution of ITS, Activin A (100 200 ng / ml) and Wnt3a (50-100 ng / ml, or approximately one day (time 0 to day 1) and one more day or in the same medium, but without Wnt3a (day 1 to day 2). they took daily cell samples for RNA isolation and analysis After 2 days of differentiation, the cultures were fed with RPMI + 0.2% FBS vol / vol containing a 1: 1000 dilution of STIs, KGF (or FGF7, 25 ng / ml) and TGFp inhibitor # 4 (2.5 pM) for one day (or 24 hours, from day 2 to day 3) During the next two days (from day 3 to day 5), the suspensions of iPS cell aggregates were fed the same growth factor cocktail media, with the exception that the TGFp inhibitor was removed from the culture media. Again, they were taken Cell samples for RNA isolation at the end of this phase (phase 2 or day 5). For phase 3 (from day 5 to day 8), the cell culture media was changed to DMEM supplement B27 at 1% vol / vol containing TTNPB [acid 4- [E-2- (5,6,7,8 -tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1-propenyl] benzoic] (3 nM), KAAD-cyclopamine (0.25 pM) and nogina (50 ng / ml). Again, cell samples were taken for RNA isolation and analysis at the end of this phase (phase 3, day 8). For phase 4 (days 8 to 14), the medium was changed to DMEM + 1% B27 supplement vol / vol containing Nogina (50 ng / ml), KGF (50 ng / ml) and Eg F (50 ng / ml ). Again, cell samples were taken for RNA isolation and analysis at the end of phase 4 (or day 14).

Real-time PCR was performed to measure the gene expression of several marker genes during the course of the differentiation. Gene expression of the specific markers or genes was first normalized to the mean expression levels of the constitutive genes, cyclophilin G and TATA binding protein (TBP) expression. The normalized relative gene expression was then shown on the bar graphs in relation to the level of expression in undifferentiated iPS cells and, therefore, represents the regulation by increase for the different markers of differentiation. In the case of OCT4, gene expression was normalized to establish the lowest sample in the data set (day 14) and, therefore, represents downward regulation during the course of differentiation.

FIG. 2A-L are bar graphs that show the relative gene expression of the identified gene (for example, Oct4, Braquiuria, Cer1, GSC, FOXA2, FOXA1, HNF6, PDX1, PTF1A, NKX6.1, NGN3 and INS) in relation to the level of expression of the same gene in undifferentiated iPS cells. The level of gene expression was normalized with respect to a set of constitutive (control) genes, and the comparison of the level of gene expression at the two different time points indicated whether there was up or down regulation for that gene or expression marker For OCT4 (FIG. 2A), gene expression was normalized and the lowest level expression sample was set to 1 (day 14). Therefore, as indicated in FIG. 2A, the relative expression levels of OCT4 represent downward regulation (Y axis) during the course of differentiation (X axis, phase 0 to 4, or day 0 to day 14 ).

As shown in FIG. 2A, OCT4 (POU5F1) is expressed at high levels in undifferentiated iPS cells and shows subsequent downregulation during the course of differentiation (from day 0 to day 14). On day 14, OCT4 expression levels were reduced by more than 3,000 times compared to the expression levels observed in undifferentiated cells. On the contrary, there was a transient upward regulation of the expression of the brachyuria gene (BRAQUIURIA, FIG.2B) during the first 2 days (day 1 and day 2). The transient upward regulation of brachyuria was the result of directed differentiation of pluripotent cells / iPS in mesendoderm by the application of Activin A and wnt3a. The mesendoderm also differed in definitive endoderm during days 2 and 3, since continued exposure to Activin A was indicated by upward regulation of CER1, GSC and FOXA2 at the end of phase 1 on day 3 (FIG. 2C-E). During phase 2, the definitive endoderm was also directed to differentiate the digestive tract endoderm as indicated by the up-regulation of FOXA1, the maintenance of FOXA2 expression and the down-regulation of CER1 and GSC on day 6 of differentiation (FIG. 2C-F). During phase 3, after exposure to retinoid, cyclopamine and nogina, the endoderm of the intestinal tract was further directed to differentiate itself to the endoderm expressing PDX1 / posterior upper digestive tract, as indicated by upward regulation of HNF6 and PDX1 in on day 8 (Figure 2G-H). During phase 4, after exposure to KGF and EGF, the posterior superior digestive tract endoderm / with PDX1 expression was further directed to differentiate into pancreatic progenitor cells, endocrine progenitors and endocrine cells expressing hormones as indicated by upward regulation PTF1A, NKX6-1, NGN3 and INS at day 14 (FIG. 2I-L).

Example 2

RHO-QUINASA INHIBITORS POWER THE GROWTH, SURVIVAL, PROLIFERATION AND ADHESION OF CELL-CELL CELL OF iPS CELLS

The methods for differentiating several cell lines from hES and iPS are essentially as described herein and in Example 1. In addition to the culture conditions described for phases 1, 2, 3, 4 and 5, apoptotic inhibitor was added and / or Rho-kinase or ROCK inhibitor to culture media to improve and enhance cell-cell growth, survival, proliferation and adhesion during differentiation. Typically, approximately 10 pM of a Rho-kinase inhibitor, for example, Y-27632, was added to the cell cultures in each of the phases. Alternatively, a Rho-kinase inhibitor was added to at least phases 1 and 2 and phases 4 and 5, or any combination thereof. The morphology and expression profiles of gene markers of differentiated iPS suspension (aggregate) cell cultures are essentially similar to those of suspension cell cultures derived from hES cells.

FIG. 3 and 4 show the immunocytochemistry (ICC) of cultures of iPS cells of phases 4 and 5, respectively. FIG. 3 shows a Phase 4 cell aggregate that expresses typical genetic markers of the pancreatic endoderm positive in PDX1 (also known as pancreatic epithelium or pancreatic progenitors) that includes positive cells in both PDX1 and NKX6.1. Although not shown in FIG. 3, Phase 4 cells do not express hormone secretory proteins or gene markers more typical of Phase 5 cells such as insulin (INS), glucagon (GCG), somatostatin (SST) and pancreatic polypeptide (PP). FIG. 4 shows the cell aggregate of cells expressing Phase 5 hormones. These ICC results were further confirmed using QPCR. However, since the QPCR is a total population study of the total level of RNA expressed in the sample or in the cell culture, it cannot be definitively demonstrated that a single cell expresses multiple markers.

Example 3

ENCAPSULATION OF PANCREATIC PROGENITORS DERIVED TO iPS

Until now, methods for the production of iPS cells and sources for the production of iPS cells have been published. However, there is not a sufficient description of the differentiation of any iPS cell to any differentiated cell that functions for potential use in a cell therapy to treat a particular disease, for example diabetes.

To determine whether cultures of PDX1-positive pancreatic endodermal cells in Phase 4 or pancreatic progenitor cells derived from human iPS cells were fully capable of developing and maturing glucose-sensitive insulin secreting cells in vivo , progenitor cell populations were loaded pancreatic essentially as described in Examples 1 and 2 in macroencapsulation devices essentially similar to those described in the US application. No. 12 / 618,659, entitled "ENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS", presented on Friday, November 13, 2009; and U.S. patents. UU. No. 7,534,608 and 7,695,965, entitled "METHODS OF PRODUCING PANCREATIC HORMONES". In summary, in each device, approximately 5-10-20 pl of gravity sedimented cell suspension aggregates, which had essentially about 3 x 10 6 cells, were loaded.

The cells encapsulated in the device were prepared for implantation in a mammal, for example, an immunocompromised SCID / Bg mouse, but can be implanted in larger animals, including rats, pigs, monkeys or human patients. The methods for implanting the encapsulated cells and the device are essentially as described in the US patent application. No. 12 / 618,659, U.S. Patent Nos. 7,534,608 and 7,695,965, including pancreatic progenitor cells implanted in a GELFOAM matrix and implanted below the fatty layer of the epididymis (EFP).

Immunosuppression was not necessary in these studies, however, immunosuppression may be necessary for certain mammals during an initial intermediate period until the parents within the device fully mature and respond to glucose. In some mammals, immunosuppression regimens can last approximately 1.2, 3, 4, 5, 6 or more weeks, and will depend on the mammal.

The transplanted cells were allowed to differentiate and mature further in vivo. To determine if the transplanted cells had a normal physiological function as a natural beta cell, for example, human insulin levels will be determined by analyzing the levels of human C-peptide. Human C-peptide is cleaved or processed from human proinsulin, therefore, the detection of specifically human C-peptide, and not endogenous mouse C-peptide, indicates that insulin secretion is derived from grafted (exogenous) cells. . Animals with implants will be tested for human C-peptide levels approximately every two, three or four weeks by injecting a bolus of arginine or glucose, preferably glucose. Then mature beta cells (derived from differentiated pluripotent iPS cells) must be physiologically functional and sensitive to glucose, unlike natural or endogenous beta cells. Generally, the amounts of human C-peptide above 50 pM or the average baseline level (threshold) is an indicator of the function of the now beta cells of the transplanted progenitors.

Similar to that described in Kroon et al. (2008) supra, the US application No. 12 / 618,659, US Pat. No. 7,534,608; 7,695,965 and 7,993,920, encapsulated pancreatic progenitor cells derived from hIPS cells are expected to mature and become functioning pancreatic islet groups that have endocrine cells, acinar and ductal cells that do not differ from those of natural islets. It is also anticipated that purified or enriched pancreatic progenitor cells derived from hIPS cells before transplantation will also mature and become functional pancreatic islets and produce insulin in vivo. Certain embodiments to purify and enrich several differentiated cell populations are described in more detail in the US application. No. 12 / 107,020, entitled "METHODS FOR PURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FROM HES CELLS", filed on Tuesday, April 8, 2008, now US Pat. No. 8,338,170. It is further anticipated that pancreatic progenitor cells derived from hIPS cells that have been cryopreserved can be thawed and adapted in culture before transplantation, and therefore mature and produce insulin in vivo. And that hypoglycemia can be improved in animals with induced diabetes that have transplanted pancreatic progenitor cells derived from hIPS cells.

In summary, fully encapsulated pancreatic progenitor cells derived from hIPS cells in a macroencapsulation device mature in physiologically functional pancreatic islets and are expected to produce insulin in response to glucose in vivo.

Example 4

COMPOSITIONS OF PANCREATIC PROGENITOR CELLS AND HORMONE SECRETORS

Differentiated hIPS cell populations were analyzed by flow cytometry to determine their content of pancreatic endodermal cells positive in PDX1 or pancreatic progenitor cells (in phase 4); and endocrine or endocrine progenitor / precursor cells (in phase 5) as shown in Tables 6a, 6b and 7, respectively. Table 6b is the same data set as in Table 6a, but presented similar to that of Table 10 for comparison. The populations of Table 6a overlap each other, for example, the total number of cells is greater than 100%, because the total numbers of PDX1 + and NKX6.1 + overlap with that of the NKX6.1 + / cell population. PDX1 + / CHGA- (5th column of Table 6a). Table 6b includes only PDX1 + and triple data negative (residual), which are not shown in Table 6a. Certain of these iPEC grafts, as well as others using essentially similar formulations, were implanted in animals to determine function in vivo ; however, the levels of C-peptide in human serum were not robust enough for a possible therapeutic purpose (data not shown). The values shown are the percentage of total cells that belong to a given population. The numbers of pancreatic progenitor cells (NKX6.1 (+) / PDX1 (+) / CromograninA (-)) and a very small population of NKX6.1 + / PDX1- / CHGA- that are in the aggregates of suspended cells coincided with those observed in suspension aggregates of pancreatic progenitor cells derived from hES cells and aggregated in the ESC phase as described in the US application. No. 12 / 264,760, entitled "STEM CELL to Gg REGATE SUSPENSIONCOMPOSITIONS AND METHODS OF DIFFERENTIATION THEREOF", presented on Tuesday, November 4, 2008. The levels of endocrine and / or endocrine progenitor / precursor cells also essentially coincided with those obtained in hES derived cell cultures in the US application. UU. No. 12 / 107.020, entitled "METHODS FOR PURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FROM HES CELLS", filed on Tuesday, April 8, 2008. Similar to suspension aggregates of cells derived from hES, the variation in concentrations of Different growth factors in the culture medium at certain stages of differentiation (for example, phase 4) should increase and / or decrease certain endodermal pancreatic, endocrine, endodermal populations positive in PDX1 or non-pancreatic cell types.

T l: m i i n l l r ni r n r i n f 4 r n l l l

T l: m i i n l l r ni r n r i n f 4 r n l l l

Table 7: m i i n l l n rin n f r n l l total)

Example 5

PEC TYPE REINFORCING KINASES

The methods described above are essentially similar to those described in the following Table 8, adapted from Schulz et al., (2012), supra. These and other methods described herein can be found in the applicant's numerous patent and non-patent publications, including US patents. UU. No. 7,964,402; 8,211,699; 8,334,138; 8.008.07; and 8,153,429.

Table 8: Conventional manufacturing method for creating pancreatic endodermal cells (PEC) derived from hESC

Phase Point Media condition Flask speed 6-time tray speed ( 1-4) rotary wells

( day) ( rpm) ( rpm)

d (-1) hESC XF HA; PU 5-10 95

Ag.

d0 1 r0.2FBS-ITS1: 5000 A100 5-10 95

W50

d1 r0.2FBS-ITS1: 5000 A100 5-10 95

d2 r0.2FBS-ITS1: 1000 K25 IV 5-10 95

d3 r0.2FBS-ITS1: 1000 K25 5-10 95

d4 r0.2FBS-ITS1: 1000 K25 5-10 105

d5 3 db-CTT3 N50 5-10 105

d6 db-CTT3 N50 5-10 105

d7 db-CTT3 N50 5-10 105

d8 4 db-N50 K50 E50 5-10 105

d9 db-N50 K50 E50 5-10 95

d10 db-N50 K50 E50 (or without 5-10 95

feeding)

d11 db-N50 K50 E50 5-10 95

d12 db-N50 K50 E50 5-10 95

hESC Ag .: hESC aggregates; XF HA: DMEM / F12 containing GlutaMAX, supplemented with 10% v / v serum replacement deactivated without Xeno, 1% v / v nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1% v penicillin / streptomycin / v (all of Life Technologies), 10 ng / ml Heregulin-1p (Peprotech) and 10 ng / ml Activina A (R&D Systems); SP: hESC SFM StemPro® (Life Technologies); r0.2FBS: Rp M i 1640 (Mediatech); 0.2% FBS (HyClone), 1 x GlutaMAX-1 (Life Technologies), 1% v / v penicillin / streptomycin; STI: Insulin-transferrinaselenium (Life Technologies) diluted to 1: 5,000 or 1: 1,000; A100: Recombinant human activin A at 100 ng / ml (R&D Systems); W50: 50 ng / ml recombinant mouse Wnt3A (R&D Systems); K25: recombinant human KGF at 25 ng / ml (R&D Systems); IV: TGF-p IV 2.5 pM RI kinase inhibitor (Em D Bioscience); db: High glucose DMEM (HyClone) supplemented with supplement B-27 x0.5 (Life Technologies), GlutaMAX x1 and 1% v / v penicillin / streptomycin; CTT3: 0.25 pM KAAD-Cyclopamine (Toronto Research Chemicals) and 3 nM TTNPB (Sigma-Aldrich); N50:

Recombinant human nogin at 50 ng / ml (R&D Systems); K50: recombinant human KGF at 50 ng / ml (R&D Systems); E50: 50 ng / ml recombinant human EGF (R&D Systems); no feeding: indicates that the cells were not fed again on the indicated day; db, DMEM (high glucose content).

When the above methods were applied to iPS cells and pancreatic progenitor cells transplanted in animals, the applicant did not systematically obtain the same consistent function in vivo compared to the application of the same methods to pancreatic progenitor cells derived from hESC and hES. This was surprising, given that iPS cells are human pluripotent stem cells that have the morphology and gene expression pattern of hESC, and can form embryoid bodies in vitro and teratomas in vivo, indicating that they can form cells of all three germ layers. See at least, for example, Yu et al. (2007); U.S. patent application publication No. 2009/0047263, International Patent Application Publication No. WO2005 / 80598; U.S. patent application publication No. 2008/0233610; International Patent Application Publication No. WO2008 / 11882, supra. These references describe that iPS cells meet the ESC definition criteria. Therefore, there is an expectation that iPS cells can replace ESCs in an in vitro differentiation protocol that produces hES-derived pancreatic progenitor cells that mature further and become fully functional glucose sensitive cells in vivo. However, given the inconsistent in vivo performance data using the above methods, applicants sought to explore a formulation of unique differentiation media for pancreatic progenitor cells and / or pancreatic endodermal cells (PEC), that is, cells derived from phase 4 of hiPSC (or "iPEC") that are capable of providing essentially similar robust levels of in vivo function that have been consistently observed for the HECP-derived PEC.

Applicants previously reported that endocrine cells (CHGA + cells) present in PEC are polyhormonal endocrine cells, and are not the subpopulation of cells in PEC that give rise to islets that have glucose-sensitive insulin secreting cells in vivo. See Kelly et. to the. (2011) supra. Rather, it is the population of non-endocrine cells (CHGA- cells), especially those that jointly express NKX6.1 and PDX-1, which are believed to be the PECs that actually give rise to the islets in operation in vivo . Therefore, the applicant explored whether the modulation, change or displacement of the relative proportions of endocrine subpopulations and non-endocrine can affect subsequent function in vivo.

Previous efforts to decipher receptor-ligand signaling in hESC successfully identified the growth factors that enhanced self-renewal and allowed the development of defined media culture conditions. See Wang et al (2007) supra. Wang et al. They identified Heregulin-1p as the ligand that bound ERBB3 and induced dimerization with ERBB2 to affect hESC self-renewal in that context. ERBB is a receptor tyrosine kinase (RTK) and RTK are transmembrane proteins that are widely expressed and act as receptors for growth factors and other extracellular signaling molecules. After ligand binding, they undergo tyrosine phosphorylation on specific residues of the cytoplasmic tail and activate a signaling cascade for the binding of other protein substrates that participate in RTK-mediated signal transduction. The role of RTK in several evolutionary processes, including the regulation of cell survival, proliferation and motility, and its role in cancer formation is well documented. It was also known that ERBB receptor tyrosine kinases were expressed throughout the developing human fetal pancreas, although the specific functions of certain ERBB receptors and their ligands are unknown. See Mari-Anne Huotari et al. (2002) "ERBB Signaling Regulates Lineage Determination of Developing Pancreatic Islet Cells in Embryonic Organ Culture", Endocrinology 143 (11): 4437-4446.

Due to the role of ERKB RTK in the self-renewal of pluripotent stem cells and their expression in the fetal human pancreas, as demonstrated by Wang et al. (2007) supra and ERBB RTK expression in the human fetal pancreas, the applicants again investigated the possible activation of RTK in in vitro pancreatic endodermal cells (PEC) derived from hESC in an effort to identify receptors and ligands that could improve the specification of PEC during differentiation, or expansion through the promotion of self-renewal, or some other unknown mechanism that potentiates the maturation of islet hormone secreting cells that function physiologically in vivo. PECs were generated in suspension in differentiating aggregates, essentially as described in Table 8, except with the following modifications.

Four PEC samples were generated for RTK transfer analysis. At the end of phase 4 (or d13), a "steady state" sample of PEC aggregates was collected in db-N50 K50 E50. A "starvation" sample represented aggregates of PEC d12 that were fed with db medium (high glucose DMEM or high glucose DMEM supplemented with Supplement B-27 x0.5 (Life Technologies)) alone (without growth factors) and were collected on d13. Two "pulsed" samples were loaded and grown on db media on d12, then on d13, loaded with db-K50 E50 medium or db medium containing 2% FBS, for 15 minutes before harvest. These conditions were intended to detect the RTKs that were active under the conditions of phase 4, and what response could be obtained with a pulse of KGF, EGF and insulin (present in supplement B27) or serum. The serum pulse was thought as stimuli of the broad-spectrum growth factor, potentially identifying the RTKs that are present in PEC and can be activated, but not stimulated with the present conditions of phase 4.

RTK analysis was performed essentially as described previously in Wang et al., (2007) supra. In summary, Proteome Profiler ™ (R&D Systems) human phospho-RTK antibody matrices were used according to the manufacturer's instructions. Protein lysates were prepared in 1% NP-40, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2.0 mM EDTA, 1.0 mM sodium orthovanadate, 10% aprotinin pg / ml and leupeptin at 10 pg / ml. 500 pg of freshly prepared protein lysates were incubated overnight with duplicate stained nitrocellulose membranes for 42 anti-RTK antibodies and 5 negative control antibodies, as well as 8 positive control stains on anti-phosphotyrosine (FlG. 5A ). Arranged antibodies capture the extracellular domains of phosphorylated and non-phosphorylated RTKs, and bound phospho-RTKs are detected with a horseradish peroxidase conjugated to horseradish peroxidase (HRP) using chemiluminescence. See FIG. 5 for the design of the matrix of r Tk, as well as Table 9 below for the list of RTK in the matrix.

Table 9: List of tyrosine kinase recept to RTK for PEC RTK analysis

(continuation)

RTK transfers (Figure 6A) indicated that insulin and IGF1 receptors (IR, IGF1R, respectively) were phosphorylated and activated in all conditions, similar to what was previously observed with hESC. See Wang et al (2007) supra. The EGF receptor (EGFR, also known as ERBB1) was phosphorylated under steady state conditions, which was expected, given the presence of EGF in the middle of phase 4. In fact, the low level phosphorylation of ERBB2 is detected both in the steady state and in the conditions of starvation. The phosphorylation of EGFR and ERBB2 was elevated in each of the pulsed conditions, confirming the ability of the assay to detect activation in response to a ligand pulse. Phosphorylated VEGFR3 was also detected in all conditions and elevated in pulsed samples. This suggested that PEC produces an endogenous VEGFR3 ligand, the possible candidates are VEGF-C and D. The serum pulse seemed to activate additional receptors, including low levels of ERBB3 phosphorylation. The detection of phosphorylated ERBB2 / 3 suggests that a member of the Heregulin-type EGF family could activate signaling in PEC. TIE-2 is one of two angiopoietin receptors, and appears to be phosphorylated at a low level in response to serum. Angiopoietin 1 and angiopoietin 4 are known to be Tie-2 activating ligands, while angiopoietin 2 and angiopoietin 3 function as competitive context-dependent antagonists. The HGF receptor (HGFR) also phosphorylated in response to serum pulse, suggesting that hepatocyte growth factor could also cause signaling in PEC. Finally, although a low level phosphorylation of the B2 RTK (EPHB2) epinephrine was detected, the efroma / Eph signaling is a cell-cell signaling system attached to the membrane, and it is unlikely that it can be easily exploited in differentiation from PEC. Interestingly, ERBB4 was not phosphorylated. Therefore, the RTK analysis highlighted several receptors that are phosphorylated in PEC, or that can be phosphorylated in response to different conditions, for example, serum. These results suggest that several soluble ligands can cause RTK signaling in PEC and may impact cell proliferation, differentiation and / or specification, and therefore may affect subsequent maturation to function in pancreatic islets in vivo.

Example 6

THE GROWTH FACTORS OF HEREGULIN AND FGF2 AFFECT THE PEC DERIVED FROM THE HESC COMPOSITIONS

In view of the RTK analyzes, which demonstrated that certain RTKs were activated (or phosphorylated) under certain conditions as described above in Example 5, and since it appeared that at least ERBB2 and ERBB3 were activated in PEC (after 13 days of differentiation of phases 1-4), the applicant tried to determine the effect of Heregulin when it was applied to cells in phases 3 and 4.

Preliminary studies were conducted with Heregulin and FGF. In some of these studies, Rho-kinase inhibitors, Y-27632 were included. These preliminary studies showed that the treatment of pluripotent stem cells during a day in phase 1 with 10 ng / ml of Heregulin-1p (the same concentration and isomer of Heregulin that is disclosed in Wang et al. (2007)) increased the size of cell aggregates derived from hES in suspension culture compared to the size of cell aggregates derived from hES in suspension culture without Heregulin-1p (Hrg1p). An increase in the size of the cell aggregate is advantageous, because it results in a greater cell mass for subsequent implantation and function test in animals. In addition, aggregate disk size increased when Hrg1p increased from 10 ng / ml to 50 ng / ml in phase 3. This result was also observed when 50 ng / ml of another growth factor, FGF2, was used in phase 3. compared to cultures in the absence of FGF2. An increase in the size of cell aggregates was also observed when phase 3 cultures were exposed to additional days of exposure to FGF2, for example, 3 days of FGF2 at 50 ng / ml compared to 2 days.

Table 10 provides a summary of the flow cytometry analysis of PEC cells treated with Hrg1p and FGF2 in phase 3. Endocrine cells are denoted as CHGA positive (or CHGA +) and non-endocrine cells are denoted as CHGA negative (or CHGA -). Endocrine (CHGA +) and non-endocrine (CHGA-) cells can be stained positively for other markers, for example, positive on PDX1 and / or NKX6.1. Cells that do not stain with any of the tested markers are called triple negative cells or residual cells (CHGA- / NKX6.1- / PDX1).

T l 1: An li iim rí fl PE riv hE rn H r lin F F2

To determine whether the increase in the size of the cell aggregates affected the PEC subpopulations, the composition of the PEC populations was analyzed by flow cytometry. Compared to the control cultures, so Hrglp and FGF2 were not used to differentiate the cells, the non-endocrine subpopulation of PEC (CHGA-) increased from 54.01% to 61.2% with the addition of 10 ng / ml of Hrglp in phase 3, and increased from 54.01% to 64.2% with the addition of 50 ng / ml of Hrg1p in phase 3. Endocrine subpopulation (CHGA +) was not significantly affected with the treatment of 10 ng / ml of Hrg1p, but even more with 50 ng / ml. In parallel, the relative levels of residual cells decreased and even more with 50 ng / ml of Hrg1p. Therefore, the increase in the size of the cell aggregates with the treatment with Hrg1p was mainly attributed to the increase in non-endocrine subpopulations in relation to endocrine and residual subpopulations.

The effect of FGF2 on phase 3 cultures was similar but even more pronounced than that of Hrg1p. For example, the PEC non-endocrine subpopulation (CHGA-) increased as it did for Hrg1p. The main effect of FGF2 in these cultures was the substantial decrease in endocrine subpopulation. In some cases, these cells were almost undetectable with 3 days of treatment (from 32.9% to 0.33%). Therefore, the increase in the size of cell aggregates for FGF2 treated cultures was mainly attributed to the increase in non-endocrine subpopulations, and in some cases, residual cell subpopulations (from 13.1% to 22.7% during 3 days in phase 3).

Therefore, Heregulin and / or FGF2 seem to play a role in the specification of cells in PEC populations. This is surprising, given that Wang et al (2007) supra reported that Heregulin alone played a role in cell renewal when used in context with pluripotent stem cells.

Example 7

METHODS FOR IMPROVING THE PEC IN VIVID INJECTION FUNCTION THROUGH THE CULTURE TREATMENT OF CELLS DERIVED FROM IPS WITH HEREGULIN

Because the methods according to Table 8 when applied to iPSC to produce iPEC did not provide a potent in vivo function in animals, applicants explored other methods for the production of iPEC. Changes in the conventional method as set forth in Table 8 include, but are not limited to: optimization of the number of times any iPSC is passed; modulation of BMP signaling levels; modulation of the aggregation parameters of the iPSC suspension during expansion and differentiation (for example, shear force, rotation speed and the like); optimization of concentrations, time of use and duration of use of growth factors, such as inhibitors of Wnt, Activin and rho-kinase; and treatment with other growth factors in various phases 1 to 4 of the differentiation protocol as candidates to improve cell mass, proliferation, differentiation, cell survival, and the like (eg, ERBB ligands). These numerous iterative experiments were tested alone or in combination, to determine how differentiation methods could be optimized for iPSCs during phases 1-4. Such optimized differentiation methods produce populations of iPEC that, when grafted, resulted in potent glucose-sensitive insulin secreting cells in vivo similar to those observed and reported for hESC. The following Table 11 describes the reference conditions, with and without Heregulin, which were demonstrated to differentiate iPSC from iPEC, which later matured to glucose sensitive islet cells in vivo. The initial conditions were similar to those described in Examples 1, 2 and 5, as well as in Table 8 of this document, except that Heregulin was added in phases 3 and 4. Although 30 ng / ml of Hrg1p were used , concentrations ranging from 10 ng / ml to 50 ng / ml, or even greater than 50 ng / ml are suitable. Also, the addition of a rhokinase inhibitor, Y-27632, was maintained in the differentiation cultures as described in Example 2.

Table 11: Comparison of the differentiation formulations of baseline and Heregulin momentum for the manufacture of pancreatic endodermal cells (PEC) derived from iPSC

Basal Moment (Without Heregulin) Phase (1-4) Basal Moment with Heregulin 20% of KSR-F10 A10 Y10 iPSC Ag. 20% of KSR-F10 A10 Y10

r0.2FBS-ITS1: 5000 A100 W100 Y10 1 r0.2FBS-ITS1: 5000 A100 W100 Y10 r0.2FBS-ITS1: 5000 A100 Y10 r0.2FBS-ITS1: 5000 A100 Y10

r0.2FBS-ITS1: 1000 IV K25 Y10 2 r0.2FBS-ITS1: 1000 IV K25 Y10

r0.2FBS-ITS1: 1000 K25 Y10 r0.2FBS-ITS1: 1000 K25 Y10

r0.2FBS-ITS1: 1000 K25 r0.2FBS-ITS1: 1000 K25

db- CTT3 N50 3 db- CTT3 N50 H30

db- CTT3 N50 db- CTT3 N50 H30

db- CTT3 N50 db- CTT3 N50 H30

db- N50 K50 E50 Y10 4 db- N50 K50 E50 H30 Y10

db- N50 K50 E50 Y10 db- N50 K50 E50 H30 Y10

db- N50 K50 E50 (or without power) db- N50 K50 E50 (or without power) db- N50 K50 E50 Y10 db- N50 K50 E50 H30 Y10

db- N50 K50 E50 Y10 db- N50 K50 E50 H30 Y10

IPSC aggregates: iPSC aggregates; KSR: serum deactivated (Life Technologies); F10: bFGF at 10 ng / ml (R&D Systems); A10: Activin A at 10 ng / ml (R&D Systems); A100: Activin A at 100 ng / ml; r0.2FBS: RPMI 1640 (Mediatech); 0.2% FBS (HyClone), 1x GlutaMAX-1 (Life Technologies), 1% v / v penicillin / streptomycin; STI: Insulin-transferrin-selenium (Life Technologies) diluted to 1: 5,000 or 1: 1,000; A100: Recombinant human activin A at 100 ng / ml (R&D Systems); K25: recombinant human KGF at 25 ng / ml (R&D Systems); CTT3: 0.25 | jM KAAD-cyclopamine (Toronto Research Chemicals) and 3 nM TTNPB (Sigma-Aldrich); N50: Recombinant human nogin at 50 ng / ml (R&D Systems); K50: recombinant human KGF at 50 ng / ml (R&D Systems); E50: 50 ng / ml recombinant human EGF (R&D Systems); Y10: Y-27632 10 jM; 20 mM stock solution, x2000; H30: Heregulin at 30 ng / ml (stock solution at 100 g / ml); db, DMEM (high glucose content).

To determine the effect of the addition of Heregulin or Heregulin and a rho-kinase inhibitor in subpopulations of phase 3 and 4 cells, iPEC populations were analyzed by flow cytometry. Table 12 provides a summary of the flow cytometry analysis of several iPEC populations using the formulations set forth in Table 11, as well as the formulations that were modified by increasing the concentration of Activin to 200 ng / ml. In addition, Table 12 shows the general conditions used for each set of experiments (baseline with or without Heregulin) and the relative percentages of the cell types of the iPEC population (endocrine, non-endocrine, PDX1 positive cell subpopulations, residual or triple negative). Table 12 also reveals data on the in vivo function of the cells produced in each experiment.

Table 12: iPEC compositions of iPS derived cell cultures treated with Here ulina

(continuation)

Under certain conditions, the ratio of cell subpopulations in the PEC (hESC, E2354) and iPEC (E2314, E2344, E2347) populations were altered. For example, sometimes, the percentage of endocrine cells (CHGA +) decreased and the percentage of non-endocrine cells (CHGA- / NKX6.1 + / PDX1 +) increased compared to the initial conditions (without Heregulin). Although it appeared that Heregulin was responsible for changing the proportions of endocrine cells in relation to non-endocrine cells in these populations of PEC and iPEC, in experiment No. 2380 (E2380), the level of endocrine cells (CHGA +) increased by instead of decreasing with the addition of Heregulin.

To determine whether the change in the composition of the PEC and iPEC populations affected function in vivo, PEC and iPEC grafts were transplanted from most of the experiments described in Table 12 to mice essentially as described previously in the This document and in other patent and non-patent publications of the applicant, including Schulz et al. (20l2) and Kroon et al (2008), supra and US patents. No. 7,534,608; 7,695,965; 7,993,920 and 8,278,106, supra. In summary, the populations of PEC and iPEC were fully encapsulated with a biodegradable semipermeable cell encapsulation device, some of which included microperforations. The devices were manufactured by the applicant, and are described in detail in US Pat. No. 8,278,106, entitled "ENCAPSULATION OF PANCREATIC CELLS FROM HUMAN PLURIPOTENT STEM CELLS", presented on Friday, November 13, 2009. Glucose-stimulated insulin secretion (GSIS) assays were performed from approximately 56 days later of the implant Blood was collected before (fasting) and in combinations 30 and / or 60 minutes after glucose administration. Graft function was evaluated by measuring the concentrations of human C-peptide in the serum in response to glucose administration.

The amount of human C-peptide released in the serum is indicative of the amount of insulin released. Peptide C is a short 31-amino acid peptide that connects or binds chains A and B of proinsulin and preproinsulin, which is secreted by the functioning of insulin secreting beta or secretory cells. As previously analyzed by Kroon et al. (2008) supra et al., Measurements of human C-peptide are appropriate for the evaluation of de novo- generated insulin release by implanted cells. Accordingly, the levels of human C-peptide in the serum of these animals are a measure of the in vivo function of mature PEC and iPEC grafts. Human C-peptide was detected in the serum at least 8 weeks after implantation. With additional weeks of implant and fasting, glucose-stimulated C-peptide levels increased with maximum levels of C-peptide displacement from 60 minutes to 30 minutes after glucose administration, which is indicative of a faster response. to glucose exposure as insulin cells mature. There were some mice that could not present the function or were sacrificed due to poor health; however, these mice were in cohorts that, on the other hand, presented high-functioning animals, thus suggesting a graft failure rather than an inability of the implanted cells to differentiate and function.

FIG. 7A-C show the levels of human C-peptide in the serum after glucose administration for all the experiments indicated in Table 12, with the exception of E2344. FIG. 7A-C show that, in comparison to the reference controls, those grafts resulting from treatment with Heregulin, in general, were found to have higher levels of human C-peptide in serum. For example, in FIG. 7A, in experiment 2380, there is an increase of approximately 5 times (933 pM: 200 pM at 60 minutes after administration of glucose) in grafts resulting from Heregulin treatment compared to preparations without Heregulin (baseline) . Heregulin appears to have a lower effect on PECs produced from hESC, since experiment 2354 (FIG. 7C) does not show higher levels of serum C-peptide in those grafts resulting from treatment with Heregulin compared to the controls of reference. In addition, when comparing the PEC derived from hESC (CyT203) and iPEC derived from iPSC, iPEC grafts have a function comparable in vivo to PEC grafts (for example, compare FIG. 7A and FiG. 7B (grafts of iPSC) with FIG.7c (CyT203 hESC) As such, iPEC grafts are as robust as PEC grafts, and the relative proportions of endocrine cells with respect to non-endocrine, which seemed to affect some of the populations of iPEC (for example, E2314, E2347 and E2354), it did not seem to affect in vivo function because iPEC of E2380, which did not have the same change in Endocrine and non-endocrine subpopulations also showed good function (see FIG.7A-C).

In addition to being tested for glucose-stimulated insulin secretion, mature iPEC grafts were tested to determine if they could only maintain euglycemia, similar to euglycemia maintained by PEC derived from hESC, if the host animal's beta cells were destroyed. this involved the destruction of the beta cells of the implanted mouse using the beta cell toxin, streptozotocin (STZ), which shows a higher cytotoxicity against murine beta cells compared to human beta cells. Blood glucose measurements were taken without random fasting for each mouse before and after STZ treatment. After implantation of the iPEC graft on day 13 after treatment with STZ, hyperglycemia was resumed (blood glucose increase should be noted), demonstrating glycemic control by the iPEC graft instead of the pancreas of endogenous mouse (see Figure 8A and Figure 8B.

In addition, there appeared to be a synergistic effect when Heregulin and a rho-kinase inhibitor were provided during phases 1-4 of differentiation (see Table 11). For example, the iPSC treated with Heregulin in phases 3 and 4 without a rho-kinase inhibitor resulted in a visibly poor cell mass, so that implantation was impossible. Additional support for the synergy of Heregulin and a rho-kinase inhibitor was evident in some of the experiments, for example, E2356, E2380, so baseline conditions with a rho-kinase inhibitor alone did not they functioned as solidly as a graft with the rhokinase and heregulin inhibitor (see Fig. 7A and B). It seems that treatment with Heregulin and a rhokinase inhibitor was not additive, because the addition of Heregulin alone provided an insufficient cell mass for transplantation, and the addition of a rho-kinase inhibitor alone (initial conditions) had a poor in vivo function. As such, the supply of Heregulin alone or a rho-kinase inhibitor alone is not essentially similar to the sum effect of the two combined. That is, by themselves, neither of them results in a robust glucose response in vivo, but in combination they produce a glucose response similar to that of hES derived cells. Therefore, it appears that the supply of Heregulin and a rho-kinase inhibitor is synergistic, since its combined effect is greater than the sum of the effect of each separately. That is, the iPEC treated with the rho-kinase inhibitor and Heregulin matured in showing glucose-stimulated insulin secretion, and were able to maintain euglycemia in a mouse model with diabetes (see FIG. 7A-B and FIG. 8A-B).

ERBB functionality requires ligand binding, receptor dimerization and receptor traffic. Variability in each process can produce differential regulation of the receptors and the down-chain signals they control. For example, different ERBB ligands bind to ERBB receptors with different affinities, therefore, altering the patterns and dynamics of ERBB dimer formation. Table 13 shows the many different possible combinations of ligands and receptor binding complexes. The reviews related to the complexity of this system are provided by Oda, et al. (2005) "A comprehensive pathway map of epidermal growth factor receptor signaling", Mol. Syst Biol., 1 (2005) and Lazzara et al. (2009) "Quantitative modeling perspectives on the ERBB system of cell regulatory processes", Experimental Cell Research 315 (4): 717-725.

T l 1: Tir in in rr ERBB li n

Huotari et al. suggested that neuregulin-4 can modulate the relative levels of endocrine cell subpopulations by increasing the number of somatostatin (delta) cells at the expense of glucagon (alpha) cells, and that neuregulin-4 did not affect the proportion of exocrine cells (e.g., amylase) to endocrine cells (e.g., p-insulin, a-glucagon, 8-somatostatin, PP pancreatic polypeptide). These studies, however, were performed by incubating neuregulin-4 in tissue cultures of whole assembly organs obtained from E12.5 day mice. These mouse explant cell populations differed beyond the populations of cells in phase 3 (for example, upper digestive tract endodermal negative in PDX1) and / or phase 4 (upper digestive tract endodermal positive in PDX1) described in This document. Neuregulin-4 only binds to ERKB4 RTK, so that only the endocrine subpopulation of the entire assembly mouse culture can be modulated by neuregulin-4 in this context. Therefore, the treatment of cells in phase 3 (upper digestive tract endodermal negative in PDX1) and / or phase 4 (upper digestive endodermal positive in PDX1) as described herein with a different ERBB ligand , for example, Hrg1, would not be expected to modulate the relative endocrine subpopulation as in Huotari, because it has already been shown that Hrg1 binds ERBB3 and induces ERBB2 / 3 dimerization. However, due to the low level expression of ERBB2 and 3 in PEC as shown in FIG. 6, it was not clear whether cells of types 3 and 4 expressed low or high levels of ERBB2 and 3 to bind to Hrg1.

In addition, in a different context, the applicant described that Hrg1 joined ERBB 2/3 and enhanced the self-renewal of pluripotent stem cells (see Wang et al (2007)). Although it is possible that Hrg1 can act in the same capacity in the context of phases 3 and 4, the applicant has previously described that most of the cell expansion for PEC production occurs in the pluripotent stem cell phase (phase 0). During phase 0, hESCs are grown, passed and expanded for approximately two (2) weeks. Therefore, most of the cell expansion or self-renewal to produce cell expansion does not occur during phases 1-4. See Schulz et al. (2012) supra. Also, assuming that ERBB2 / 3 is present during phases 3 and 4, heregulin could be expected to have the same effect as with pluripotent stem cells (self-renewal) instead of having an impact on directed differentiation. The difference in function seems to depend on context, that is, pluripotent stem cells versus a type of endodermal or pancreatic lineage cell.

In summary, the supply of Heregulin or Heregulin and a rho-kinase inhibitor in vitro to upper digestive tract endodermal cells (phase 3) and pancreatic endodermal cells expressing PDX1 (end of phase 3 and phase 4) produced populations of p Ec and iPEC, which when transplanted, matured and developed in glucose-sensitive insulin secretory cells in vivo (see FlG. 7 and 8). This use of Heregulin or Heregulin and a rho-kinase inhibitor has been reported herein for the first time. Such use and effect are not discernible from what was previously described in the patent or non-patent literature.

Example 8

THE ACTIVINA SUPPRESSES THE EXPRESSION OF NGN3 AND THE PRODUCTION OF CELLS COMMITTED TO THE ENDOCRINE LINEAR IN CROPS OF ENDODERMAL CELLS (PEC) PANCREATICS

Pancreatic endodermal cells, or "PEC", are a pancreatic population of cells that are mainly composed of two distinct subpopulations: (i) a multipotent non-endocrine pancreatic progenitor subpopulation (CHGA-) (hereafter, "non-endocrine (CHGA) -) "), which develops and matures giving rise to glucose-sensitive insulin-secreting beta cells in vivo ; and (ii) a subpopulation of cells compromised with the endocrine lineage (CHGA +) (hereafter referred to as "endocrine (CHGA +)"), which may result in other cells that do not secrete insulin or maintain islets during maturation in alive. The non-endocrine subpopulation (CHGA-) is negative in CHGA and predominantly positive in PDX1 and NKX6.1, while the subpopulation of cells compromised with the endocrine lineage is positive in CHGA and, mostly, negative in PDX1 and NKX6.1 . Non-endocrine subpopulation (CHGA-) takes approximately 8 to 12 weeks or more to develop and mature to glucose-sensitive insulin-secreting beta cells in amounts that allow systemic detection of insulin responses (as analyzed by ELISA for peptide C) in vivo. It takes, however, less than 8 weeks for glucose sensitive cells to develop and mature in vivo during normal embryonic development. Therefore, it is still desirable to obtain (1) a population of PEC with an increase in non-endocrine subpopulation (CHGA-) and / or (2) a true population of endocrine cells (CHGA +) that is glucose sensitive in vitro and / or that it is an evolutionarily advanced population (for example, adequately specified and with certain distinctive markers consistent with those described for similar pancreatic populations in vivo) and / or (3) a population of progenitor cells that is capable of shortening and / or reduce the duration of the period of development and maturation in vivo.

With respect to the first objective, it is desirable to obtain a population of PEC, whereby the non-endocrine subpopulation (CHGA-) remains intact while, at the same time, differentiation to cells committed to the endocrine lineage (CHGA +) is repressed in a manner similar to what occurs naturally in vivo. The following examples describe methods for the production of said PEC population.

During normal development in vivo, it is believed that Neurogenin-3 (NGN3), a transcription factor, is the main regulator of endocrine cell development, and that NGN3 expression does not arise until after regulation upward of PDX1 and NKX6.1, for example, in pancreatic endodermal lineage cells in vivo. See Rukstalis and Habener (2009), "Neurogenin3: A master regulator of pancreatic islet differentiation and regeneration", Islets 1 (3): 177 184. According to Table 8, in phase 3, retinoic acid (RA) is used or the retinoid analog, 4- [(E) -2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1-propenyl] benzoic acid or "TTNPB "(or" TT3 "in Table 8) to induce PDX1 expression. However, at the same time, retinoic acid also prematurely induces the expression of NGN3 that initiates the endocrine specification. Therefore, the suppression, repression or inhibition of NGN3 during phase 3 and / or phase 4 can provide a clue to minimize cells compromised with endocrine lineage subpopulation (CHGA +) in PEC and / or obtain endocrine cells in later phases of in vitro and / or in vivo development. Importantly, any suppressor, repressor or inhibitor of NGN3 should not simultaneously suppress, suppress or inhibit the non-endocrine subpopulation (CHGA-) and preferably this subpopulation increases during the in vitro production of PEC.

Various growth factors were explored for their ability to suppress genes such as NGN3 during phases 3 and 4. A candidate growth factor was Activin, which has various functions in the expansion and differentiation of stem cells. For example, at low levels (for example, 10 ng / ml), Activin helps maintain the pluripotence of pluripotent stem cells and cell proliferation. At higher levels (for example, 100 ng / ml), Activin acts as a growth factor by differentiation responsible for the production of definitive endoderm in phase 1 (see, for example, Table 8). To test its effect on the repression of cells compromised with endocrine lineage (CHGA +) during PEC formation, the applicant first tested Activin at a medium level concentration of approximately 50 ng / ml with conventional adherent differentiation. At this level, Activin was able to suppress the expression of NGN3 without suppressing the expression of the PDX1 and NKX6.1 genes in PEC (compare FIG. 9D-E with FIG. 9A-B). However, when the same level of Activin was used with a more scalable cell aggregate suspension culture during phase 3, the overall cell mass decreased dramatically and, in some cases, cell aggregates were removed (compare control with aggregates Cells treated with Activin in Figure 10). Similar decreases were observed in cell aggregates for Activin at 25 ng / ml as with 50 ng / ml (data not shown).

In view of the results, the lowest concentrations of Activin (for example, 5 ng / ml and 10 ng / ml) were tested in phases 3 and 4. As shown in FIG. 11, at lower concentrations of Activin, cell aggregates were able to maintain cell mass and performance compared to the control (compare FIG. 11B-C on day 8 and FIG. 11B-D on day 12 with FIG. 11A, control, for d8 and d12). Therefore, Activin levels below about 25 ng / ml are acceptable to maintain cell mass and performance. The applicant then performed a flow cytometry analysis on these cell cultures to determine if Activin at low levels during phases 3 and 4 had any effect on the composition of the cell aggregate; see Table 14. Table 14 shows that low levels of Activin do not decrease the production of non-endocrine subpopulation (CHGA-), and when the treatment is applied in phases 3 and 4, in fact, it can increase this subpopulation ( see Table 14 A5 Phase 3 and 4 results). However, it seems that differentiation to cells compromised with endocrine lineage (CHGA +) is still being significantly induced given the very similar percentages of endocrine cells (CHGA +) observed with and without Activin (see the endocrine column in the Table 13). Therefore, although Activin can suppress or inhibit the expression of NGN3 and NKX2.2 as observed with QPCR, at lower levels (which allows to maintain cell mass) it does not reduce the subpopulation compromised with the endocrine lineage (CHGA +) when it is analyzed at the end of phase 4. Advantageously, the negative subpopulations only in PDX1 and Triple Negatives were reduced compared to the control, in particular when the Activin is used in phases 3 and 4.

Example 9

ACTIVINA, HEREGULINA AND WNT SUPPRESS THE PRODUCTION OF COMMITTED CELLS WITH THE ENDOCRINE LINE IN PEC CROPS

Example 8 showed that moderate levels of Activin (for example, 50 ng / ml) during phases 3 and 4 were able to suppress or repress the expression of NGN3 (FIG. 9), but that these same levels did not maintain cell mass . The lowest levels of Activin (for example, 5 and 10 ng / ml) were able to maintain cell mass (Figure 11), but were not sufficient to continue suppressing endocrine differentiation during phase 4, since the percentage of cells of the endocrine lineage (CHGA +) remained essentially similar to the control (Table 14, CHGA + column) and to that described in the previous examples. Therefore, it seems that the suppression of the differentiation of cells committed to the endocrine lineage (CHGA +) mainly during phase 3 at these low levels is not sufficient, because the continued suppression of this subpopulation through the phase is still considered necessary. 4. See Rukstalis et al. (2009) supra. Therefore, additional growth factors that were able to prevent cell loss and performance were explored (i.e., counteract the effect of moderate to high levels of Activin), while at the same time not affecting production of multipotent non-endocrine pancreatic progenitor subpopulations and suppressed the differentiation of cells compromised with the endocrine lineage (CHGA +).

In view of the above examples describing that Heregulin at 30 ng / ml during phases 3 and 4 usually changes the relative levels of endocrine (CHGA +) and non-endocrine (CHGA-) PEC subpopulations, the applicant tested what effects it could have the reduction of Heregulin concentrations when combined with Activin in the production of PEC.

Pluripotent stem cells were differentiated using the conventional protocol according to Table 8, except that, during phase 3, Activin was included at 10 ng / ml or 20 ng / ml in combination with Heregulin at 2 ng / ml or 10 ng / ml; and, during phase 4, the Activin was reduced to 5 ng / ml with Heregulin at 1 ng / ml for two days and then to 10 ng / ml thereafter. As shown in FIG. 12 (images from day 10; upper panel), Activin at 10 ng / ml and Heregulin at 2 ng / ml showed some cell loss compared to the control (compare FIG. 12A and B, upper panel). Increasing Activin to 20 ng / ml with Heregulin that remains equal to 2 ng / ml aggravated cell loss (compare FIG. 12B and C, upper panel). However, prevention of increased cell loss at higher concentrations of Activin (20 ng / ml) was possible by increasing Heregulin to 10 ng / ml in the context of Activin to 20 ng / ml (compare Figure 12 D and C, top panel). Therefore, when heregulin levels are too low, a significant loss of cells is observed, but when heregulin levels rise, for example, at least 20 ng / ml, there is a change in the percentages of non-endocrine cells (CHGA-) compared to the subpopulations of the endocrine lineage (CHGA +).

A quantitative PCR analysis was performed to determine the relative levels of gene expression on days 8, 10 and 12 of differentiation with the previous combinations of Activin and Heregulin (FIG. 13). Similar to what is shown in Example 8 (Fig. 9), Activin at 10 and 20 ng / ml was able to repress NGN3 and NKX2.2 (another endocrine differentiation marker) without decreasing NKX6 expression. 1 (compare FIG. 13A and B with that of C). The fact that the expression of NKX6.1 remains high indicates that the production of non-endocrine subpopulations (CHGA-) remains unchanged or unaffected. Specifically, however, at 20 ng / ml of Activin and 10 ng / ml of Heregulin, or 10 ng / ml of Activin and 2 ng / ml of Heregulin, the Activin-dependent cell loss that was previously observed when used at 25 ng / ml and 50 ng / ml (FIG. 10) was greatly reduced (FIG. 12 D and E, upper panel). Therefore, Heregulin seems to effectively counteract the cell loss originally observed by the use of Activin in phase 3. Also, although some induction of NGN3 and NKX2.2 still occurs (that is, it is not as repressed as observed when 50 ng / ml of Activin were used in phases 3 and 4 in the differentiation based on adhesion in Example 8, FIG. 9), it is significantly reduced compared to conventional conditions (compare control conditions and conditions A20H10 of FIG. 13A and B).

Although more than 20 ng / ml of Activin would further repress the NGN3 and NKX2.2 markers, and therefore, would further repress endocrine differentiation, then higher levels of Heregulin would be required to maintain cell mass in order to compensate for the Effect of the dose of Activin at high concentrations. However, higher levels of Heregulin such as those observed in the previous examples may change the relative production of endocrine (CHGA +) and non-endocrine (CHGA-) subpopulations of PEC. Even so, the effect of higher levels of Activin in repressing the expression of NGN3 remained desirable. Therefore, applicants explored additional growth factors or morphogens that may affect the fates of the anterior and posterior endoderm, and control other aspects of development, including, but not limited to, WNT, FGF, PDGF, retinoic acid (RA or TTNPB) , BMP, hedgehog, EGF, IGF and the like.

In particular, the involvement of the canonical Wnt signaling pathways was explored by the addition of Wnt3A in the differentiation media of Activin and Heregulin in phases 3 and / or 4. Again, any combination must suppress cell differentiation. compromised with the endocrine lineage (CHGA +) observed by repressing the expression of NGN3 and NKX2.2, and maintaining a good cell mass (i.e., a limited cell mass loss), while at the same time not affecting to multipotent pancreatic progenitor subpopulation not endocrine One of the mixtures analyzed was 50 ng / ml of Wnt together with 10 ng / ml of Activin and 2 ng / ml of Heregulin in phase 3; and as before, during phase 4, Activin at 5 ng / ml with Heregulin at 1 ng / ml for two (2) days, then Activin at 5 ng / ml and Heregulin at 10 ng / ml later. This gave a greater cell mass (PEC) than Activin and Heregulin alone at the same concentrations (compare FIG. 12D and E, upper panel), and even a greater cell mass than the control (compare FIG. 12A and E, top panel). The QPCR analysis of cell aggregates under these conditions (for example, combination of Activin, Heregulin and Wnt in phase 3, and Activin and Heregulin in phase 4) showed that there was a significant suppression of cells compromised with lineage differentiation endocrine (CHGA +) observed by decreased expression of NGN3 and NKX2.2 in phases 3 and 4, in particular on days 8, 10 and 12 (FIG. 13 A and B). However, although the expression of NKX6.1 was initially repressed on day 8, it rose significantly on days 10 and 12 (phase 4) (FIG. 13C). Therefore, the development of non-endocrine subpopulation (CHGA-) was not affected with the addition of Wnt, and the differentiation of cells committed to the endocrine lineage (CHGA +) seemed to be delayed or suppressed, and there was no apparent loss. of cell mass.

To determine the cellular composition of the PEC population with the combination of Activin, Heregulin and Wnt during phase 3, a flow cytometry was performed after phase 4, and the cellular composition of PEC is shown in Table 14. As shown, the combined growth factors (Activin, Heregulin and Wnt, or "AHW") effectively reduced the differentiation of cells committed to the endocrine lineage (CHGA +) as observed by the decreased percentage of this subpopulation (15, 6 vs. 58.8) while increasing non-endocrine subpopulation (CHGA -) - (58.1 vs. 23.4) compared to control cell cultures under the conventional protocol (Table 8). The flow cytometry data supports and coincides with the QPCR data in that the combination of the three factors (AHW) in general delayed or suppressed the differentiation of cells compromised with the endocrine lineage (CHGA +) in PEC without affecting the non-endocrine subpopulation production (CHGA-). Delayed expression of these genes in vitro coincides with their onset (or delay) in the pancreatic development of mammals in vivo, so that the expression of NGN3 in endocrine differentiation occurs after the start of PDX1 and NKX6.1 expression in non-endocrine subpopulation (CHGA-). In addition, there was no compromise in mass or PEC performance.

In view of the results obtained using the combination of Activin, Heregulin and Wnt, the levels of Activin were adjusted again in phase 3 to establish a range of concentrations in which Activin could be used. The concentrations of Activin tested varied from approximately 0.25, 50, 75 and 100 ng / ml with Wnt at 50 ng / ml and Heregulin at 5 ng / ml during phase 3. The Activin was reduced to 5 ng / ml, Heregulin at 5 ng / ml and Wnt was not included during phase 4. The cells were photographed at the end of phase 4 (day 13) and are shown in FIG. 12 (bottom panel). Cell loss was again evident at higher concentrations of Activin, for example, between 75 and 100 ng / ml (FIG. 12D and E, lower panel). Importantly, the inclusion of WNT in phase 3 allowed the use of a much higher concentration of Activin in phase 3 than would have been possible due to cell loss.

To test the effect of Activin and Heregulin continued during phase 4, in the context of Activin, Heregulin and Wnt in phase 3, human ESCs were differentiated using three conditions: (i) conventional protocol (see Table 8; o Protocol No. 1 of Table 17); (ii) combination of Activin (75 ng / ml), Heregulin (5 ng / ml) and Wnt (50 ng / ml) in phase 3, followed by the conventional factors of phase 4; and (iii) combination of Activin (75 ng / ml), Heregulin (5 ng / ml) and Wnt (50 ng / ml) in phase 3, followed by more Activin (5 ng / ml) and Heregulin (5 ng / ml) during phase 4, see Table 17. Flow cytometry was performed after phase 4 for all three conditions, and the cellular composition of PEC is shown in Table 17. The AHW in phase 3 decreases the cells committed to the endocrine lineage and increases the content of non-endocrine subpopulations (CHGA-) compared to the control (21.8 vs. 42.2 for cells compromised with the subpopulation of the endocrine lineage; and 40.1 versus 27, 6 for non-endocrine subpopulation (CHGA-), and further treatment with AH in phase 4 further decreases cells compromised with endocrine lineage and further increases the content of non-endocrine subpopulation (CHGA-) (6.46 versus to 21.8 for cells compromised with the endocrine lineage; and 72.7 versus 40.1 for the non-endogenous progenitor subpopulation Crina (CHGA-).

Table 16: Activin, Heregulin and Wnt in phases 3 and 4 decreases endocrine subpopulations and increases CHGA- non-endocrine sub oblations

Example 10

PEC GRAFTES PRODUCED USING THE COMBINATION OF ACTIVINE, HEREGULIN AND WNT HAVE IMPROVED THE FUNCTION SENSITIVE TO GLUCOSE IN VIVO

Example 9 showed that based on morphology data (eg, low-power microscopy images of differentiated cell aggregates), QPCR and flow cytometry, it appears that the combination of Activin, Heregulin and Wnt ("AHW") ) can effectively suppress cells compromised with endocrine lineage differentiation (CHGA +) observed by repression of NGN3 and NKX2.2 expression, maintain or increase non-endocrine subpopulations (CHGA-) while, at the same time, have no no detrimental effect or improvement in cell mass or performance. To determine whether these changes have an effect on the final development and maturation of glucose-sensitive insulin secretory cells, PWA aggregates derived from animals were implanted in animals, together with the PECs created with the conventional protocol in accordance with the Table 8 or Protocol # 1 of Table 17.

Specifically, human pluripotent stem cells were differentiated using the combination of the three factors (AHW) in phase 3, and then Activin and Heregulin alone in phase 4. The PEC control was performed essentially in accordance with Table 8. Cellular aggregates were analyzed by QPCR on days 8, 10, 12 and 14 as shown in FIG. 14. Both the control and the modified AHW protocol produced PEC as observed by the NKX6.1 expression (Figure 14A), although there is a more potent NKX6.1 expression using AHW on day 14 (after phase 4 ). At the same time, the differentiation of cells committed to the endocrine lineage (CHGA +) was largely suppressed with the AHW protocol as observed by repression of NGN3 expression, especially on day 8 compared to the control protocol ( FIG. 14B).

The PEC aggregates of both methods were loaded into the implantable semipermeable encapsulation devices patented by the applicant on day 14 of differentiation and implanted in beige SCID mice essentially as described above (n = 5 animals for each of the AHW and control methods, n = 10 in total). At 11 weeks after implantation, PECs produced using the AHW protocol turned out to be an average of approximately 900 pM C-peptide after one hour (60 ') of glucose exposure, while the control PECs were approximately 400 pM C-peptide. See FIG. 15, in which the animals of AHW are compared with those of the control. Specifically, at 11 weeks after implantation, 3 of the 5 mice with AHW implants averaged more than 1,200 pM of human C-peptide (FIG. 15, animals # 3514, 3516 and 3518), while peptide levels C in control animals were significantly lower. In fact, only 1 of the 5 control animals turned out to have levels close to 600 pM (FIG. 15, animal No. 3524). Therefore, based on C-peptide levels, at 11 weeks after implantation, the AHW protocol was able to produce a population of PEC with at least twice the improvement in function in vivo, measured according to the peptide level Human C after glucose stimulation. This potentially correlates with a faster maturation time.

At 14 weeks after implantation, PEC AHW implants continued to outperform control graft implants, and with a faster insulin secretion response time, because C-peptide values 30 minutes after exposure to the glucose for PW AHW implants were higher than 60 minutes after exposure for FIG control implants. 16). Specifically, 3 of the 4 animals with PEC AHW implants averaged more than 3,500 pM C-peptide 30 minutes after glucose exposure, compared with the first 3 control animals that averaged approximately 2,300 pM but after 60 minutes after exposure to glucose (FIG. 16). This data demonstrates that differentiation using the combination of the three growth factors (AHW) is capable of producing a better functioning PEC population (i.e., increased levels of serum C-peptide 30 minutes after glucose exposure, which indicates a faster glucose response of PEC grafts derived from implanted AHW) or is potentially more potent than the conventional PEC protocol at the time points studied. Without limiting this application to any theory, it is believed that the increase in potency in AHW PEC implants is attributed to the fact that the combination of AHW (1) delayed or repressed the differentiation of cells compromised with the endocrine lineage ( CHGA +) in PEC; (2) increased subpopulation of multipotent non-endocrine pancreatic progenitor cells in PEC; and (3) maintained a good cell mass.

Example 11

IN VITRO PANCREATIC ENDOCRINE CELL CULTURE

Examples 9 and 10 describe the deliberate delay, repression, suppression or inhibition of differentiation of cells committed to endocrine lineage (CHGA +) in phases 3 and 4. In an effort to produce an evolutionarily endocrine cell precursor cell advanced or an endocrine cell in vitro, applicants tested various methods during phases 5, 6 and / or 7. For example, the applicant tested which growth factors, alone or in combination, were able to stimulate or induce endocrine differentiation, that is, induce the expression of NGN3.

The following Table 17 summarizes several methods for endocrine production together with the conventional protocol for producing PEC (Protocol No. 1) and the protocol used to produce PEC AHW (Protocol No. 2). Table 17, Protocol No. 3, indicates a series of growth factors in specific days and phases, however, applicants have proven, and one skilled in the art will appreciate that not all growth factors, alone or in combination, have to be used in the exact day / phase indicated. Therefore, Table 17 represents the numerous iterations (for example, growth factors, duration and the like) tested by applicants.

T l 17: M rr ir ll PE n rin in vi r

(continuation)

hESC Ag .: hESC aggregates; XF HA: DMEM / F12 containing GlutaMAX, supplemented with 10% v / v serum replacement deactivated without Xeno, 1% v / v nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1% v penicillin / streptomycin / v (all from Life Technologies), Heregulin-1b at 10 ng / ml (Peprotech) and Activin A at 10 ng / ml (R&D Systems); SP: hESC SFM StemPro® (Life Technologies); r0.2FBS: RPMI 1640 (Mediatech); 0.2% FBS (HyClone), 1x GlutaMAX-1 (Life Technologies), 1% v / v penicillin / streptomycin; cb: c Mr L: CMRL 1066, Glutamax x1, 1% v / v penicillin / streptomycin, 2% B-27; db: DMEm Eli Glucose (HyClone) supplemented with a supplement B-27 x0.5 (Life Technologies), GlutaMAX x1 and 1% v / v penicillin / streptomycin; A100, A50, A5: 100 ng / ml, 50 ng / ml, recombinant human Activin A at 5 ng / ml (R&D Systems); BMP20, BMP10: 20 ng / ml, BMP4 at 10 ng / ml (Peprotech); CTT3: 0.25 mM KAAD-cyclopamine (Toronto Research Chemicals) and 3 nM TTNPB (Sigma-Aldrich); E50: 50 ng / ml recombinant human EGF (R&D Systems); H10, H5: 10 ng / ml, Heregulin 1b at 5 ng / ml; IGF25: IGF2 at 25 ng / ml (Peprotech); STI: Insulin-transferrin-selenium (Life Technologies) diluted to 1: 5,000 or 1: 1,000; IV: RI kinase IV inhibitor of 2.5 mM TGF-b (EMD Bioscience); K50, K25: 50 ng / ml, recombinant human KGF (continuation)

at 25 ng / ml (R&D Systems or Peprotech); MG0.05: 0.05 % MATRIGEL (BD Biosciences); N50: Recombinant human nogin at 50 ng / ml (R&D Systems); NC10: 10 mM nicotinamide; PDGF10: Platelet-derived growth factor at 10 ng / ml (Pd Gf, R&D Systems); RO1: gamma-secretase inhibitors, RO4929097, 1 mM; SHH100: sonic hedgehog at 100 ng / ml; TTNPB1: TTNPB 1 nM (Sigma-Aldrich); W50: 50 ng / ml recombinant mouse Wnt3A (R&D Systems); Y10: Y-27632 10 mM (Tocris Bioscience).

Previously, the use described by the applicant of a gamma secretase inhibitor for the production of endocrine progenitor / precursor cells in US Pat. UU. No. 8,129,182, "ENDOCRINE PROGENITOR / PRECURSOR CELLS, PANCREATIC HORMONE EXPRESSING CELLS AND METHODS OF PRODUCTION", granted on Tuesday, March 6, 2012. As described and claimed in patent 182, to further induce differentiation of the positive pancreatic endodermal cells in PDX1 towards the endocrine lineage, growth factors or agents that inhibited Notch signaling were elucidated. This was achieved by the application of a gamma secretase inhibitor (for example, DAPT or N- [N- (3,5-difluoropheacetyl) -L-alanyl] -S-phenylglycine t-butyl ester and related analogs). Gamma-secretase inhibitors block intramembrane cleavage of the Notch molecule, thus preventing the release of the activated Notch intracellular domain. A 2 to 4 day application of the DAPT gamma secretase inhibitor was used, for example, on the final days of retinoic acid addition or immediately after retinoic acid extraction to induce NGN3 expression. In this case, another gamma-secretase inhibitor, RO44929097 was used during phase 5. See Table 17.

As indicated in Table 17, in order to promote the differentiation and / or maturation of the endocrine lineage, during phases 6 and 7, a combination of growth factors was applied that included, but are not limited to, BMP4 ("BMP"), Sonic Hedgehog ("SHH"), platelet-derived growth factor ("PDGF"), FGF2 ("FGF"), retinoic acid and / or retinoic acid analogs, such as TTNPB ("TTNPB"), growth factor of type insulin I (IGF-I or "IGF1") and II (IGF-II or "IGF2"), a derivative of vitamin B3, Nicotinamide ("NC") or combinations of one or more of these growth factors. One such combination is described in Table 17, protocol No. 3.

For example, the PEC prepared from the AHW protocol essentially as described above in Example 10 was further treated in phase 5 (days 13 and 14) with the gamma secretase inhibitor, RO4929097 ("R01"), normally , for 2 days at 1 pM, according to Table 17, Protocol No. 3. This phase 5 treatment produces the induction of NGN3 expression and, subsequently, endocrine differentiation. In addition, nicotinamide was included during phases 5 and 6 to also induce NGN3 expression or endocrine differentiation. The expression of the NGN3 gene was analyzed by Nanostring detection of mRNA after phase 4 (cultures of day 13), after phase 5 (cultures of day 15) and after 2 days of phase 6. As shown in FIG. 17, the gamma-secretase inhibitor strongly induced NGN3 expression after phase 5 on day 15 compared to low level expression of NGN3 at the end of phase 4 (day 13). After removal of the gamma-secretase inhibitor on day 15 (first day of phase 6), the expression of NGN3 decreases as observed on day 17 (FIG. 17). This transient increase in the expression of NGN3 during phase 5 coincides with its transient expression observed during the development of beta cells in vivo. See Jorgensen et al. (2007) supra.

It is noted that the total number of days for any of the phases may vary and varies, for example, phases 5, 6 and / or 7 may be shortened or lengthened in the number of days. This also applies to the previous phases 1, 2, 3 and 4.

In addition, a rho-kinase inhibitor can be added to the differentiation media continuously or intermittently during any of phases 1, 2, 3, 4, 5, 6 and 7 to enhance cell survival, cell mass and performance A rho-kinase inhibitor in phases 1, 2 and 4, for example, seems to enhance the differentiation of pluripotent stem cells induced, for example, by maintaining cell mass and performance.

Also, on approximately day 20 (i.e., the junction between phases 6 and 7 of Protocol # 3 of Table 17), cell aggregates can be dissociated and added again. This dissociation and reintegration eliminated certain cellular subpopulations, thus leaving a population of predominantly endocrine and / or endocrine / progenitor / progenitor (CHGA +) precursor cells. This physical manipulation also facilitates the analysis of endocrine populations and constitutes a stage of purification, if needed. For more information, see Example 14.

In addition, starting from phase 6 to phase 7, if necessary, a very low concentration, for example, approximately 0.05%, of Matrigel can be used to help maintain cell aggregates and / or prevent endocrine cells migrate from cell aggregates. The use of Matrigel or any other complex cell matrix may depend on the specific cell aggregates themselves (eg, PEC aggregates, endocrine progenitor / precursor cell aggregates as described below, and the like) and, potentially, on the PSC line. from which cell aggregates were derived. It is possible to use other complex matrices or matrices defined, for example, serum, laminin, fibronectin, collagen and other extracellular matrix proteins, to perform essentially similar functions. For more information, see Example 14.

The addition of insulin-like growth factor (IGF) 1 and / or 2 (IGF1 or IGF2) can be added in phase 6 or 7 or during phase 6, for example, from day 16, 17 or 18 Pluripotent stem cells differed essentially as described above in accordance with Table 17, except for the addition of IGF1 at 50 or 200 ng / ml or IGF2 at 25 or 100 ng / ml to cell cultures during phase 6, specifically on day 17 for about 3 days. Cellular aggregates were analyzed for mRNA using Nanostring after phase 6 (day 20). These preliminary studies showed that IGF2 proved to be more capable of inducing INS expression than IGF1 (data not shown); however, a longer treatment could produce a more potent effect of IGF2 alone or IGF1, or the combination of the two.

Table 17 describes the use of various growth factors at certain concentrations; however, the invention is not limited to the specific concentrations described herein, since one skilled in the art will recognize that the modification of the growth factor concentration is conventional in the art and, in fact, is described in the Previous examples with at least changes in the levels of Activin and Heregulin, and their effect on cell differentiation and composition. Modifications herein include, but are not limited to, 5, 10, 20, 50 to 100 ng / ml of Activin; 5, 25, 50 to 75 ng / ml of Wnt; 2, 5, 10, 30 ng / ml Heregulin; sonic hedge-hog a 10 to 200 ng / ml (SHH); from 10 to 100 ng / ml of PDGF; from 10 to 20 ng / ml BMP; from 2 to 20 ng / ml of FGF2; and from 25 to 200 ng / ml of IGF1 and / or IGF2. In addition, any of the combinations of these factors can be used, and the phase or period in which they are used, even significantly, can be varied without departing from the described invention.

Example 12

IN VIVO FUNCTION OF PANCREATIC ENDOCRINE CELLS

Until now, enriched populations of endocrine cells produced in vitro from PSC have not resulted in glucose-sensitive insulin-secreting beta cells in vivo. Therefore, the applicants tested whether the above methods according to Table 17 and those described in Example 11 above could produce the first glucose-sensitive in vitro endocrine cell in vivo.

Human pluripotent stem cells were differentiated according to Protocol No. 3 of Table 17 for phases 1-5; however, in phases 6 and 7, only FBS, Matrigel and a rho-kinase inhibitor were used, in addition to the base DMEM medium with B-27 supplement. RNA expression was analyzed by Nanostring, with time points at the beginning of phases 5 (d13) and 6 (d15), and at the end of phases 6 (d20) and 7 (d26). FIG. 18A-D and 19A-D show relative levels of mRNA expression for markers of multipotent non-endocrine pancreatic progenitor subpopulations, including PDX1, NKX6.1, SOX9 and PTF1A, and endocrine cell markers including insulin (INS), glucagon (GCG ), somatostatin (SST) and ghrelin (GHRL). At the end of phase 4 (day 13), PDX1, SOX9, NKX6.1 and PTF1A were highly expressed. All these markers are reduced during phase 5 as endocrine differentiation or induction occurs (day 15). Also, at the end of phase 6 (day 20) or at the beginning of phase 7, the mRNA levels of PDX1, NKX6.1 and PTF1A have decreased, at the same time as the mRNA levels of INS, GCG, GHRL and SST increased (compare FIG. 18A-D and FIG. 19A-D on day 20). Specifically, the decrease in the expression of PDX1 and PFT1A, and the drastic concomitant increase in the relative expression of mRNA of INS, GCG, GHRL and SST through phases 5 and 6 are due to the combination of Activin, Heregulin and Wnt (AHW), and in particular, to Activin, in phases 3 and 4. That is, the inclusion of Activin in phases 3 and 4 suppressed endocrine differentiation in phases 3 and 4, while gamma-secretase in phase 5 induced endocrine differentiation in phases 5, 6 and 7. Similar levels of NKX6.1 expression on days 20 and 26 (phases 6 and 7) compared to the previous time points shown coincided with the expression of this marker in pancreatic endocrine cells, as well as in multipotent non-endocrine pancreatic progenitor subpopulations. Therefore, in later phases (for example, phases 5, 6 and 7), the cells were directed towards endocrine differentiation, while they were suppressed from doing the same in the previous phases, phases 3 and 4.

After phase 6, the cell aggregates were dissociated and added again (described in more detail below in Example 14), which eliminated some types of cells created during phases 3 and 4, including certain subpopulations of PEC, for example, non-endocrine cells (CHGA-). The elimination of non-endocrine subpopulations (CHGA-) and / or exocrine cells and / or exocrine progenitor cells and / or duct progenitor cells and / or cells can be seen by the drastic decrease in SOX9 and PTF1A after aggregation (FIG .18, compare day 20 with day 26). After phase 7, endocrine cells were loaded into devices on day 26 and transplanted to RAG2 mice on day 27 essentially as previously described for transplantation (or implantation) in beige SCID mice. FIG. 20 shows serum levels of human C-peptides after fasting and one hour (60 ') after administration of or exposure to glucose. At 10 weeks after implantation, the five animals were producing insulin as observed by human serum P-peptide. At 15 weeks after implantation (FIG. 21), all animals showed increased levels of insulin production compared to 10 weeks after implantation, and 2 of the 5 animals averaged 1500 pM C-peptide, which is evidence of a potent ability to respond to glucose in vivo.

Before loading the endocrine cells into devices on day 26, samples were taken to verify that functional beta cells developed, matured and emerged from transplanted endocrine cells of phases 6 and 7 and not, for example, from any multipotent pancreatic progenitor subpopulation no endocrine remaining. Immunocytochemistry was performed using endocrine and non-endocrine markers in the samples. The frozen sections of day 26 were sewn with antibodies using DAPI (FIG. 22A), NKX6.1 and chromogranin A (FIG. 22B). It is clear that the vast majority of positive (red) cells in NKX6.1 also stain positively for chromogranin (CHGA +), an endocrine marker. Therefore, it appears that non-endocrine multipotent pancreatic progenitor cells, as shown by the lack of CHGA- / NKX6.1 + cells, do not appear to be present in any significant number in these endocrine cell preparations.

Samples of these same endocrine cell aggregates were also stained together for INS, NKX6.1 and PDX1 (FIG. 23A-B). FIG. 23A-B showed that most insulin-expressing cells also express NKX6.1 and PDX1. This is a surprising result given that previously published reports of cells expressing insulin derived in vitro from PSC do not jointly express NKX6.1 and / or PDX1. See US patent. u No. 7,033,831 and related applications of Geron Corporation and Jiang et al. (2007), "Generation of insulinproducing islet-like clusters from human embryonic stem cells", Stem Cells, August 2007; 25 (8): 1940-53. Epub May 17, 2007.

Together, these data provide convincing evidence that, for the first time, an endocrine cell culture derived from PSC in vitro can (1) jointly express INS, Pd x 1 and NKX6.1 in vitro, and is likely to be sensitive to glucose in vitro; and (2) can lead to glucose sensitive insulin secreting cells in vivo.

Example 13

RETINOIC ACID, NICOTINAMIDE AND BMP INCREASE THE EXPRESSION OF PDX1 AND NKX6.1 IN ENDOCRINE CELLS

A hallmark of mature beta cells is that they regulate upward NKX6.1 and PDX1, at the individual cellular level, in relation to the levels observed in non-endocrine subpopulations (CHGA-) of PEC. Examples 11 and 12 described the production of endocrine cells, whereby most of the cells have NKX6.1 and PDX1 expression levels similar to those observed for non-endocrine subpopulation (CHGA-) of PEC. Therefore, it is desirable to identify growth factors that specifically improve the expression of at least these two markers in insulin-positive endocrine cells.

Two candidate factors include TTNPB, a retinoic acid or retinoid analog, and BMP4, a member of the family of bone morphogenetic proteins that is part of the superfamily of transforming growth factors beta. TTNPB and BMP4 were tested in various combinations in phases 6 and 7, and in various concentrations, but usually at 0.5 nM and 10 ng / ml, respectively. As shown by Nanostring mRNA analysis in FIG. 24, BMP in phases 6 and 7, only in phase 7, or in combination with TTNPB, can simultaneously increase the expression of insulin and PDX1 (FIG. 24A and C). On the contrary, TTNPB only in these same phases (phases 6 and 7 and phase 7 alone) does not seem to increase the expression of PDX1, and even seems to slightly decrease the expression of PDX1 compared to BMP alone (compare columns 4 and 5 of FIG. 24C with columns 2 and 3). The effects of BMP on the expression of NKX6.1 are less evident, since, in all conditions, (treatment with BMP alone, TTNPB alone, BMP and TTNPB combined, treatment of each in phases 6 and 7, or only in phase 7), The expression of NKX6.1 is relatively stable. As an additional indicator of the specificity of BMP, it was also observed that BMP upregulated the expression of the differentiation inhibitor (ID) gene (for example, ID1), which is known to be regulated by members of the TGF superfamily -p, including BMP4 (FIG. 24D). Therefore, without limiting such application to any theory, it seems that BMP is the factor that selectively induces upward regulation of PDX1 and iD1.

To determine if some of these markers were expressed together in any cell, the endocrine cell aggregates were fixed on day 27 (after phase 7) of differentiation, and frozen sections were prepared and stained by immunocytochemistry (ICC) for peptide C, NKX6.1 and PDX1. See FIG. 25 28. In this case, Instead of staining with INS, the cells were stained with an antibody against peptide C, a central fragment of proinsulin, and therefore, this staining is also indicative of cells expressing insulin (FIG. 25). The results of ICC coincided with the previous Nanostring data, that is, that BMP alone, or combined with TTNPB, in phases 6 and 7, or only in phase 7, was able to induce PDX1 expression in cells expressing insulin (peptide C) (Fig. 25 and 26).

Despite the fact that mRNA data suggested that BMP and TTNPB alone or in combination had little effect on NKX6.1 expression, ICC studies showed that, at the cellular level, the joint expression of NKX6-expressing cells. 1 and peptide C (or insulin) is quite potent (Fig. 27 and 28), that is, the staining of NKX6.1 is bright, and is also usually brighter than that observed in the multipotent non-endocrine pancreatic progenitor subpopulation. Therefore, the expression of NKX6.1 seems to be expressed predominantly together with cells expressing C-peptide (or insulin). The combined mRNA (Nanostring) and protein (ICC) data also demonstrate that, in certain contexts, there may be a limit to using only one methodology to characterize initially a type of cell. Therefore, the ICC data demonstrated that BMP alone or in combination with TTNPB in phases 6 and 7, or only in 7, was able to increase the levels of PDX1 and NKX6.1 in cells expressing C (or insulin) peptide ) (FIG. 27 and 28). The strongest effect is observed when the two factors are used in combination, but BMP alone is able to achieve the same. TTNPB can also regulate independently of PDX1 and NKX6.1. For example, treatment with TTNPB for less days, for example, 2, 3 or 4 days during phase 7 was able to induce the expression of NKX6.1 without the presence of BMP (data not shown). Therefore, the use of these growth factors, in particular, for TTNPB, may be time dependent.

Referring specifically to nicotinamide, FIG. 29 shows that nicotinamide under the culture conditions of phase 6 increases INS expression and decreases GCG expression, resulting in a triple increase in the INS / GCG expression ratio. The role of nicotinamide is unclear, but nicotinamide has been reported to prevent the loss of beta cells in rodents as a result of streptozotocin treatment, by inhibiting PARP-1, poly (adenosine triphosphate [ADP] -ribose) polymerase -1, and preventing the depletion of NAD +. See Masiello, P. et al. (1998) "Experimental N iDd M Development of a New Model in Adult Rats Administered Streptozotocin and Nicotinamide", Diabetes, 47 (2): 224-9. Nicotinamide can also increase endocrine differentiation and insulin content in human fetal islets. See Otonkoski T. et al. (1993), "Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells". J Clin Invest 92: 1459-1466.

Again, mature beta cells in vivo express high levels of PDX1 and NKX6.1, and high levels of PDX1 and NKX6.1 are necessary for proper function. Therefore, BMP, and BMP in combination with TTNPB, by inducing high expression of both markers in insulin-positive cells indicates that, for the first time, glucose-sensitive endocrine cells can be produced not only in vivo but also potentially in vitro.

Also, as mentioned above, the days for phases 6 and 7 are not rigid, and can be shortened, or even lengthened, and a person skilled in the art will recognize this, since the 5 and 10 days for phases 6 and 7, respectively, they can be easily manipulated; in particular, if essentially the same growth factors are used as described herein and in accordance with Table 17.

Example 14

MATRIGEL ONLY AND IN COMBINATION WITH A RHO KINASE INHIBITOR IMPROVES CELLULAR ADHESION OF ENDOCRINE CELL AND PEC AGGREGATES

In Example 11 above, cell aggregates were dissociated and re-aggregated or re-associated between phases 6 and 7. Dissociation was performed with Accutase on or around day 20 and added again in a rotation culture. , essentially as described in Schulz et al. supra. The reintegration removes certain subpopulations of PEC, including the remaining non-endocrine subpopulations (CHGA-), as well as other non-endocrine cells, as described in more detail in Kelly et al. (2011), supra. Endocrine cells have an affinity with each other, however, this in vitro interaction is relatively weak, even cells reacgregated in phases 6/7 were only loosely associated. Therefore, studies were conducted to test various factors and agents to determine their ability to increase cell-to-cell interactions within endocrine cell aggregates.

It was found that the addition of a Y-27632, a diluted rho-kinase and Matrigel inhibitor (0.05% v / v) was able to enhance closer cell associations of the aggregates of re-aggregated endocrine cells. The combination of Y-27632 and Matrigel increased at least, for example, cell-to-cell interaction of cell aggregates against any of the components alone.

A differentiated culture produced essentially as described in Example 12, on day 20, was dissociated with Accutase and added again in db with 5% FBS (the lowest concentrations of FBS, i.e. 2% or 1% They are also effective) and DNAse. Y-27632 was not included in the reintegration medium, since doing so would have resulted in the incorporation of non-endocrine subpopulations (CHGA-), as well as other non-endocrine cells in the newly formed aggregates. The next day, and every day after, cell aggregates were treated with Y-27632 or Matrigel, or with both components. Images were taken with a stereomicroscope 1, 2 and 3 days after aggregation, as shown in FIG. 30. FIG. 30 demonstrates that the combination of Matrigel, Y-27632 and db-FBS was the most effective. This combination was more effective than using db-FBS alone or db-FBS and Matrigel or db-FBS and Y-27632 (compare FIG. 30A-D). The cell aggregates using the combined agents seemed morphologically more adjusted for the rest of the other samples (FIG. 30D). Agents are likely to improve cell-to-cell contact and interactions between cells and the matrix; although subsequent experiments in the absence of FBS, and only Matrigel and Y-27632 gave the same results. These tighter cell aggregates appear more morphologically as organic tissue. This is advantageous so that cell aggregates can better withstand the stresses of the rotational culture, loading on devices, manipulation for transplantation, have improved development and maturation in vitro and in vivo, and the like.

Example 15

THE ABSENCE OF NOGINA PROVIDES PEC ENRICHED IN THE MULTIPOTENT PROGENITING SUB-POPULATION DOES NOT ENDOCRINE WITH LIMITED ENDOCRINE CELL CONTENT AND WITHOUT THE NEED OF FRACTIONATION OR PURIFICATION OF THE PEC POPULATION

As discussed above, the production of a PEC population (phase 4) that is highly enriched in the non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-), but relatively replete with cells compromised with the endocrine lineage (CHGA +), is desired. Such cell populations could, for example, be useful for detecting molecules or conditions that specifically induce their differentiation to endocrine lineage cells. Compounds with this activity may be useful for regenerative medicine applications to generate endocrine cells, including pancreatic beta cells either in vivo or ex vivo. Since it is known that fractionation and purification of specific cell populations from more complex cell mixtures lead to a loss of efficacy, due to cell death and for other reasons, it is beneficial to be able to create highly enriched cell populations in a phenotype desired through effective directed differentiation alone, without the need, therefore, of purification steps.

Undifferentiated human ESCs were expanded and differentiated essentially as described above in Table 17, Protocol # 1 for phases 1-4 and in Schulz et al. (2012) supra, except that during the three (3) days of phase 3, 4 different conditions were compared: (i) 0 nogina ("no nogina") during the 3 days; (ii) 1 day of Nogina at 50 ng / ml and 2 days without additional Nogina; (iii) 2 days of Nogina at 50 ng / ml and 1 day without additional nogina; and (iv) 3 days of Nogina at 50 ng / ml. After phase 3, each of these conditions (i) to (iv) received the conventional phase 4 differentiation media cocktail according to Table 8 and Table 17, but without additional Nogina. Flow cytometry analysis after phase 4 showed that Nogina produced a multipotent non-endocrine pancreatic progenitor subpopulation greater than 90% with less than 5% of cells compromised with the endocrine lineage (CHGA +). See Table 17. On the other hand, with each additional day of treatment with nogina, fewer multipotent non-endocrine pancreatic progenitor subpopulations occurred (compare 90.6% without nogina with 64.6% with 3 days of treatment with nogina) . On the contrary, Each additional day of treatment with nogina increased the production levels of cells compromised with the endocrine lineage (CHGA +).

TABLE 18: Longer duration of the effects of Nogina on subpopulations of endocrine to non-endocrine PEC cells

The QPCR analysis of cell cultures after phase 4 (day 12) showed that, with each additional day of treatment with Nogina, there was an increase in the expression levels of the endocrine lineage marker, including NKX2.2 and INS ( FIG. 31B and D), which coincides with the flow cytometry data of Table 18. On the contrary, the level of expression of the marker genes for the multipotent non-endocrine pancreatic progenitor subpopulation, including NKX6.1 (and PDX1), was relatively abundant during any period of time of treatment with nogina.

Example 16

AGENTS THAT MODULATE THE EXPRESSION OF PANCREATIC LINEAGE GENES IN PHASE 7

Next, it was evaluated whether there were additional modulators of the pancreatic lineage gene expression. Differentiation methods essentially similar to those described in Examples 13 and 14 (ie, AHW in phase 3, AH in phase 4, gamma secretase and rho-kinase inhibitors in phase 5, Matrigel e rhokinase inhibitor in phases 6 and 7). In addition, other agents were tested to determine their ability to modulate gene expression including BMP, Nogina, Fg F1, FGF2, FGF7, EGF, Hrg, HGF, SCF or TTNPB on days 30-35, during phase 7. Expression RNA was analyzed by Nanostring at the end of an extended phase 7 (d35). FIG.

32A-C show that BMP increases the expression of INS, PDX1 and ID1, coinciding with the data shown in FIG. 24 and described in detail in Example 13. BMP also decreased GHRL expression. FGF1 (acidic FGF) upregulated GCG and SST. Surprisingly, FGF2 on days 30-35 of phase 7 produced a significant increase in the expression of SST with the concomitant inhibition or suppression of the other hormone genes (eg, INS, GCG, GHRL; FIG. 32B, panel SST).

These studies demonstrate that certain agents can still modulate and affect gene expression and, therefore, differentiation of cell culture and the resulting cell type of these more differentiated / compromised cells.

Example 17

CELLULAR CROPS IN DISSOCIATION OR REAGRAGATION REDUCE NON-ENDOCRINE SUBPOBLATIONS (CHGA-) AND ADEQUATELY INCREASE ENDOCRINE SUBPOBLATIONS (CHGA +)

Previously, it has been observed that dissociation and aggregation of cell cultures can effectively deplete or reduce the presence of certain subpopulations of cells when they are added again and, as a consequence, re-aggregation can be used to enrich certain subpopulations of cells. Studies were designed to determine the effects, if any, of cell cultures regrouped with their enriched subpopulations, in this case, endocrine subpopulations (CHGA +). (see Example 14).

The differentiation methods were essentially similar to those described in Example 14, including dissociation and reintegration of cellular aggregates derived from hES before phase 7 (d20). Figure 33A-C shows RNA analysis after phase 7 (around d29) showing that non-endocrine subpopulations (CHGA-) decreased significantly in regrouped cell cultures as evidenced by Nanostring analysis using a combination of markers which include PTF1A, SOX9, PDX1 and NKX6.1 (FIG. 33A). Figure 33B also shows that aggregation resulted in an increase in pancreatic and endocrine islet cell markers, including INS, GCG, pancreatic polypeptide (PPY), GHRL, member 8 of the solute vehicle family 30 (SLC30A8 ), Glucose-6-phosphatase 2 (G6PC2), prohormone convertase 1 (PCSK1) and glucokinase (GCK).

Alone, these markers are observed in other cell types, for example, PTF1A is also expressed in exocrine cells, SOX9, in ductal cells, PDX1 and NKX6.1, in beta cells. Therefore, the applicants obtained data from several PE, endocrine and islet markers as a reference to determine the significance of any marker expression. Likewise, although four PE and non-endocrine (CHGA-) markers (PTF1A, s OX9, PDX1 and NKX6.1) were tested, only subpopulations of PTF1A and SOX9 were depleted of cell cultures regrouped at the end of phase 7 ( around d25, d26, d27, d28, d29, d30 and more). In contrast, the expression of PDX1 and NKX6.1, which is also observed in endocrine beta and pancreatic cells, remained high after phase 7. See FIG. 33A.

Similarly, other endocrine markers (for example, beta and alpha cells) that include SLC30A8, G6PC2, PCSK1 and GCK were observed at higher expression levels in d20 cell cultures re-compared compared to those cultures that were not dissociated and reunited (or "not reag."). See document FIG.

33C. SLC30A8, for example, is a zinc transporter related to insulin secretion, and certain SL30A8 alleles may increase the risk of developing type 2 diabetes; Glucose-6-phosphatase 2 (G6PC2) is an enzyme found in pancreatic islets; prohormone convertase 1 (PCSK1), together with PCSK2, processes insulin proinsulin in pancreatic islets; and, finally, glucokinase (GCK) facilitates phosphorylation of glucose to glucose-6-phosphate and is expressed in the cells of the liver, pancreas, intestine and brain, where it regulates carbohydrate metabolism by acting as a glucose sensor , triggering changes in cellular metabolism or function in response to increasing or decreasing glucose levels, such as occurs after a meal or fasting. GCK mutations can cause unusual forms of diabetes or hypoglycemia. Increasing the expression of these markers in phase 7 indicates that dissociation and reintegration of cell cultures effectively deplete or reduce non-endocrine subpopulations (CHGA-) and increase endocrine subpopulations (CHGA +). Therefore, Applicants have now perfected The further a method that produces endocrine cells in vitro properly specified for in vivo function.

Flow cytometry analysis of cell cultures in phases 1-7 also confirms that dissociation and reintegration of cell cultures as previously increases total endocrine subpopulations (CHGA + cell enrichment) with concomitant decrease in non-endocrine subpopulations. (CHGA) -o subpopulations of PE cells. See the following Table 19.

Table 19: The re-aggregated cell cultures increase the total endocrine subpopulations (CHGA +) and decrease in the non-endocrine CHGA sub-oblations after Phase 7

Until now, functional beta cells can only be differentiated through an in vivo transplant phase , and an authentic functional beta cell completely in vitro has not yet been described . See Zhou, et al. (2008), Nature 455, 627-632. Therefore, for the first time, applicants have demonstrated a population of endocrine cells (CHGA +) in vitro greater than 50% that have less than 10% non-endocrine cells (CHIGA-) and that are properly specified. Likewise, said cells are capable of developing and maturing in authentic functional beta cells and islets in vivo; See the following Example 18.

Example 18

IN VIVO INSULIN PRODUCTION FROM ENDOCRINE SUB-POPULATIONS (CHGA +)

As a follow-up study of Example 17, studies were designed to determine the effects in vivo, if any, of cell cultures regrouped with their enriched endocrine subpopulations (CHGA +).

Cultures of human pluripotent stem cells were expanded and differentiated essentially similar to that described above in Examples 12-14 and 16 and in Schulz et al. (2012), supra, except that, approximately at the beginning of phase 7 (approximately d20), the cultures were grouped and separated into 2 samples: 1) the cell aggregates were dissociated and added again ("re-aggregated cell culture"); and 2) cell aggregates that were not dissociated and added again (the control). In addition, both samples also included BMP, RA or an analogue of RA, TTNPB, and a 0.05% rho-kinase and Matrigel inhibitor during phase 7 (cell cultures from d20 to d28). Before the samples were transplanted, samples were taken for flow cytometry and Nanostring analysis (Table 19 above). Table 19 shows that the culture of cells reacgregated at approximately d29 contained approximately 11% more endocrine subpopulations (CHGA +) than the control. Importantly, the re-aggregated sample contained 9.25% less non-endocrine (CHGA-) or PE cells (only 1.45%) than samples that were not added again.

The two samples were loaded separately and encapsulated in the applicant's semipermeable, biocompatible, ENCAPTRA® EN20 ™ drug delivery devices (an animal-sized research device), and were transplanted subcutaneously essentially as described in Example 10 above in a total of 10 Beige SCID mice (5 animals received a reattached cell culture; 5 animals received control cell cultures).

Figure 34 shows after 21 weeks of transplantation, or in life, the fasting human C-peptide serum levels (the first of the three bars on the left for each animal), thirty minutes (30 '; second bar) and a hour (60 '; third bar) after administration of or exposure to glucose. At 21 weeks of the implant, all but two animals (Animal # 4317 and 4323; one in each cohort) produced potent insulin levels as observed mostly above 932 to 2691 pM C-peptide in human serum . However, when comparing the stimulation rates between the two groups (the difference between the serum level of C-peptide at 30 'or 60' and the level of fasting serum C-peptide) there was no significant difference in function. in vivo between aggregate cell cultures and control cohorts. Therefore, if the transplanted cultures or grafts consisted of approximately 98% of cultures of endocrine / re-aggregated CHGA + cells or approximately 87% of endocrine / CHGA + (Control) cells, both appear to have an in vivo function . Therefore, applicants have demonstrated for the first time that a population of in vitro endocrine cells (CHGA +) (a properly specified endocrine cell) can lead to physiologically functional insulin secreting cells when implanted.

Therefore, for the first time, applicants have demonstrated an in vitro population of properly specified endocrine cells, which, when transplanted, develop and mature to functional cells of the pancreatic islets that secrete insulin in response to blood glucose . In addition, the in vivo function is similar to that described above by the applicant for p Ec and published in Schulz et al. (2012), supra. In addition, it appears that provided that properly specified endocrine cells have been manufactured by the methods described herein through phases 1-7, additional enrichment of endocrine cells (or depletion of non-endocrine cells) through Dissociation and aggregation is not necessary or required for in vivo function . These aspects of the invention have never been demonstrated so far. To date, applicants and others only demonstrated in vivo function using a pancreatic progenitor cell population, and not a population of endocrine cells. In fact, as described in Kelly et al. (2011) supra, it is shown that endocrine subpopulations (CHGA +) of PEC or pancreatic progenitor cell preparations did not give rise to function in vivo .

Example 19

COMPLEX BASED MEANS FOR THE GROWTH OF ENDOCRINE CELLS

Complex base media, such as CMRL and RMPI, are widely used to grow islet cells and preserve the islet cell mass. CMRL media are usually more complex (more components or ingredients) than RPMI. In addition, both CMRL and RMPI are more complex than DMEM, a means of common use and less complex for cell culture. Therefore, applicants explored whether such complex base media would be beneficial for maintaining endocrine cells during the last stages of differentiation, for example, phases 6 and 7.

Islet transplantation requires the cultivation and grafting of islets with an adequate mass of islet cells and minimal toxicity. Short-term islet culture has been linked to rapid degradation and loss of viability and glucose control. See, S. Matsumoto, et al. (2003), "A comparative evaluation of culture conditions for short-term maintenance (<24 hr.) of human islets isolated using the Edmonton protocol ", Cell Tissue Banking, 4, p. 85; and NJM London, et al. (1998)," Isolation, Culture and Functional Evaluation of Islets of Langerhans ", Diabetes & Metabolism, 23, 200-207. In 1978, a study by Andersson et al. (1978) compared the efficacy of TCM, RPMI, CMRL, DMEM and Hams F10 for the cultivation of mouse islets. See Andersson A. (1978) "Isolated mouse pancreatic islets in culture: Effects of serum and different culture media on the insulin production of the islets", Diabetelogia, 14, 397 404. Andersson's results showed that Ham's F10 provided cultures of islets with the highest insulin content, but that RMPI provided cultures with the best insulin biosynthesis rate, and that this was possibly due to the high level of glucose and nicotinamide in the media.More other researchers, such as Davalli et al. (1992) showed that TCM with adenosine phosphate and xanti na were better for the cultivation of porcine islets than CMRL and RMPI. See Davalli, AM et al. (1992), "In vitro function of adult pig islets: Effect of culture in different media", Transplantation, 60, 854-860. Davalii et al. They also suggested that the high glucose level found in Ham's F10 and RMPI was possibly toxic to porcine islets. Therefore, islets of different species may have different culture requirements, and there is no suitable formula for all endocrine and islet cells. See Andersson (1978), p. 4 above

The following Table 20 describes only those components found in the CMRL, RPMI and DMEM media that vary between each of the media types, that is, Table 20 does not list the shared components in the 3 base media types, if not that only those components that are contained in CMRL but not in RPMI or / or DMEM appear. Table 20 shows that CMRL is the most complex (the largest number of components) and DMEM is the least complex (the least number of components). S, indicates the presence of that component in that base medium; and N, indicates that the component is not present in the media. In summary, DMEM lacks approximately 7 components (N, shaded boxes) that are present in CMRL and RPMI.

Table 20: CMRL RPMI DMEM cell culture media comparison

(continuation)

The methods of preparing endocrine cell cultures were essentially similar to those described above, except that, during phase 6 (d15), or the middle phase 7 (d23), or the middle phase 7 (d26), or phases 6 and 7 (d15 to d25), DMEM or CMRL base media were used. In addition, some cultures received insulin such as growth factor (IGF), nicotinamide (NC), glucose (Glc) and / or a rho-kinase inhibitor (Y-27632).

In one experiment, phase 7 cells, beginning around d23, were cultured in CMRL or DMEM base media with supplement B27, Matrigel, BMP, IGF and Y-27632. Nanostring analysis of d26 samples (phase 7) showed that, in general, phase 7 cells cultured in CMRL had increased non-specific gene expression of both hormonal and pancreatic endocrine markers compared to cells in the same phase when they were They grew in DMEM. See FIG. 35A-C. Thus, CMRL may be important for the maintenance of endocrine cells.

The corresponding studies were also performed using RPMI compared to DMEM and CMRL based media (data not shown). The CMRL and RMPI media had similar effects on cell cultures, and both improved with respect to DMEM, therefore, components that are not present in DMEM and are present in both CMRL and RPMI may be important for the maintenance of endocrine cells during phase 6 and / or 7, including reduced glutathione, amino acids, hydroxyl-L-proline, L-proline, L-aspartic acid, L-glutamic acid and vitamins, biotin and para-aminobenzoic acid.

Example 20

CRIOCONERVATION DOES NOT REDUCE ENDOCRINE CELL NUMBERS

A viable cell therapy in the market will require a larger-scale manufacture of the cellular product and a means to store the product in the long term according to the needs demanded by the patients. Therefore, any cellular product, whether single or encapsulated cells, such as the ENCAPTRA® drug delivery device, may need to be cryopreserved or have other means for long-term storage without damaging or toxic effects to the cells; and without affecting the therapeutic effect of the cells or, in this case, the in vivo production of insulin in response to blood glucose. Therefore, applicants investigated whether phase 7 cryopreserved endocrine cells maintain their ability to develop and function in vivo in a manner similar to that observed for freshly prepared (non-cryopreserved) and cryopreserved PECs as described in detail in US Pat. . UU. No. 8,278,106 and 8,425,928, both entitled, "ENCAPSULATION OF PANCREATIC CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS", issued on October 2, 2012 and April 23, 2013, respectively; and US request No. 61 / 775,480, entitled "CRYOPRESERVATION, HIBERNATION AND ROOM TERMPERATURE STORAGE OF ENCAPSULATED PANCREATIC ENDODERM CELL AGGREGATES", filed on March 7, 2013, and Kroon et al (2008) supra.

The methods of preparing endocrine cell cultures were essentially similar to those described above, except that the cell cultures were all in CMRL-based media during phase 7 and were divided into the following samples: 1) Not dissociated or regrouped, but cryopreserved approximately on day 23 for several hours and then thawed and cultured for several days in CMRL base medium with supplement of B27, BMP, TTNPB, and a rho-kinase inhibitor (Not reag./cryopreserved); 2) Not dissociated or reintegrated and not cryopreserved (Not reag./ Not cryopreserved); and 3) Dissociated and reintegrated and not cryopreserved (Reag./Not cryopreserved). The above 3 samples were grown under the same conditions as the media of phase 7. The samples were analyzed by flow cytometry on day 27. See Table 21 below.

Table 21: Flow cytometry analysis of cryopreserved endocrine cells, freshly prepared and reacted from phase 7.

Based on the flow cytometry analysis of Table 21, for those cultures that were not dissociated or rearranged (samples 1 and 2), the cryopreservation of the cells (sample 2) does not reduce the total number of endocrine cells (CHGA +) in comparison with freshly prepared cells (sample 1) (88.57 vs. 89.97). As expected, the total endocrine population (CHGA +) is higher in crops that have dissociated and regrouped, and not cryopreserved (92,85). Therefore, dissociation and reintegration affect endocrine cell numbers (CHGA +), as described above in Example 17, but cryopreservation of phase 7 cultures does not appear to alter cell composition.

In addition, the samples were analyzed using cellular immunohistochemistry with staining for NKX6.1, C-peptide, INS and GCG. See FIG. 36A-B. Endocrine cell aggregates (CHGA +) of phase 7 (Non-reag./Not cryopreserved; sample 2) showed joint expression or joint staining of NKX6.1 (nuclear) and C-peptide (cytoplasmic) (FIG. 36A-C). No joint staining was observed in similar PEC analyzes of a phase 4 differentiation protocol. Phase 7 cell cultures were also mainly hormonal, as can be seen in FIG. 36A-C, which shows the separate staining of cells with INS (36A; cytoplasmic) and GCG (36B; cytoplasmic), that is, most of the individual cells did not jointly express INS and g Cg as observed with endocrine subpopulations (CHGA + ) of PEC of phase 4. FIG. 37 shows photomicrographs with similar staining patterns, but of cell cultures that had dissociated and regrouped at the beginning of phase 7.

Figure 37 compares the graft function of cell cultures 1 and 3 of Table 20 above. Analysis of the in vivo function of the transplanted phase 7 grafts of both animal cohorts at 12 weeks showed that the cultures that had been cryopreserved but had not been regrouped (sample 1) had serum C-peptide levels similar 30 and 60 minutes after exposure to glucose compared to those cultures that had not been cryopreserved, but that had regrouped (sample 3). These levels of peptide C are comparable to those observed for weeks 10 and 15 in Fig. 20 and 21 (for example, at week 15, the average level of peptide C was 995 pM at sixty minutes after administration of glucose). Therefore, it is expected and anticipated that a subsequent analysis will show that C-peptide levels will increase above 1,000 pM at 30 and / or 60 minutes, in most animals, of glucose stimulation similar to that described in Example 18 and observed in FIG. 34. In fact, animal # 4490, a graft from Sample 1, had serum C-peptide levels at 12 weeks that were comparable to those of animal # 4320 of Example 18 (Figure 34) after 21 weeks of implant.

Therefore, phase 7 endocrine cells in cryopreservation do not appear to affect their function in vivo .

Example 21

THE INCREASE OF GLUCOSE CONCENTRATIONS AFFECTS THE EXPRESSION OF HORMONES

Glucose levels in cell culture formulations vary from 1 g / l (5.5 mM) to 10 g / l (55 mM), and some media have approximately 5.5 mM glucose, which approximates normal blood sugar levels in vivo. Glucose levels that approach 10 mM are prediabetic levels, and those above 10 mM are analogous to a diabetic condition. In other words, glucose above 10 mM mimics hyperglycemic conditions in vivo. The high glucose level can cause, for example, secondary post-translational modifications, including glycation, glyoxidation and carbonyl stress. Since reports of the effects of high glucose levels on different cell types in vitro vary, applicants sought to study the effects of high glucose levels (i.e. 4.5 g / l or 24.98 mM glucose) on endocrine cells in phase 6 and / or 7.

Glucose is a soluble hexose sugar that is added to all cell culture media, including Ames Medium; Eagle Basal Medium (BME); Fitton-Jackson modification of the BGJb medium; Click medium; CMRL-1066 medium; Eagle medium modified by Dulbecco (DMEM); DMEM / Ham F-12 Nutrient Blend (50:50); Modification of Coon F-12; Fischer's medium; H-Y medium (Hybri-Max®); Dulbecco medium modified by Iscove (IMDM); McCoy Modified Medium 5A; MCDB medium; Medium 199; Eagle's Minimum Minimum Medium (EMEM); NCTC medium; Nutrient mixture, Ham F-10; Nutrient Blend, Ham F-12; Kaighn Modification of Ham F-12 Nutrient Blend (F12K); RPMI-1640; Hybridoma medium without serum / without proteins; Half Waymouth MB; Williams E medium and several patented media. The L-15 medium contains galactose instead of glucose. See "Sigma-Aldrich Media Expert", available online at sigmaaldrich.com/life-science/cell-culture/learning-center/mediaexpert/ glucose.html.

Media containing glucose supplement levels greater than 10 mM include at least DMEM / Ham F-12 nutrient mixture (50:50) containing 17.5 mM glucose; DMEM (high glucose level), GMEM and IMDM contain glucose levels of 25 mM; and the HY medium (Hybri-Max®) and the hybridoma medium without serum / without proteins contain 22.6 and 28.9 mM glucose, respectively. See "Sigma-Aldrich Media Expert", available online at sigmaaldrich.com/life-science/cell-culture/learning-center/media-expert/glucose.html. Because the different cell culture media contain very different levels of glucose, and the effects of glucose in general vary from one cell culture system to the next, the effects of high glucose levels on endocrine cells were studied.

Again, the methods of preparing endocrine cell cultures were essentially similar to those described above, except during phases 6 and / or 7, cell cultures were treated with CMRL base medium with and without high glucose content (concentration of total glucose 4.5 g / l, or 25 mM). Therefore, this is 3.5 g / l in addition to the 1,000 mg / l or 1 g / l (5.5 mM) glucose already found in CMRL media. Both conditions with and without exogenous glucose were treated with the same growth factors, including nicotinamide, BMP, RA (or TTNPB) and, alternatively, a rho-kinase and Matrigel inhibitor. Nanostring analysis showed that, when a high glucose content was added at the beginning of phase 6, at the end of phase 6 there was a higher expression of INS, GCG and SST, and a decrease in GHRL expression (FIG. 39A and B). Therefore, the increase in exogenous glucose during phases 6 and 7, with the exception of Ghrelin, increases hormonal expression.

Example 22

IMMATURE BETA CELL PRODUCTION

Examples 8-21 describe various iterative methods for the production of endocrine cells in vitro, including the use of high level of Activin alone or in combination with Wnt and / or Heregulin in phase 3; low level of Activin in phase 4 alone or in combination with Heregulin; Nogin and a gamma secretase inhibitor with or without KGF, EGF and a rho-kinase inhibitor in phase 5; and one or more of the following between nicotinamide, retinoic acid, BMP and Matrigel in phases 6-7. The populations of endocrine cells produced from such methods are not of a single hormone (for example, INS only, GCG only or SST only); see FIG. 39), but also jointly express other immature endocrine cell markers, including NKX6.1 and PDX1.

A flow cytometry analysis was performed on two different phase 7 cultures using INS, SST and GCG hormone staining (data set A and data set B). The data set (A) was performed after examining several iterations of phases 1-7, and the other data set (B) was performed earlier in time, for example, in the absence of nicotinamide, BMP, CMRL and the like . See the following Table 22. Table 22 shows the total percentages of positive staining in INS, SST and GCG (see 5 columns on the left), total negative staining on iNs, SST and GCG (see 5 columns on the right) and staining of a single hormone (see the central column). The hormonal staining of these two data sets (A and B) is compared with that obtained from PEC phase 4 cultures that had been maintained in culture until approximately day 26.

Table 22: Comparison of cells expressing Phase 7 and Phase 4 hormones (PEC)

Flow cytometry of hormones (A) of Phase 7 (d27)

Phase 7 hormone flow cytometry (B) (d29)

(continuation)

Phase 7 hormone flow cytometry (B) (d29)

Hormone flow cytometry (PEC) in Phase 4 (d26)

In general, the total positive subpopulation in INS (the sum of only positive cells for more positive INS together for more positive INS / SST together for more positive INS / GCG together for INS / SST / GCG) is as much as triple in cultures Endocrine in phase 7 compared to PEC cultures in phase 4 (49.3 vs. 22.7 vs. 15.8). Therefore, the total subpopulation of INS alone (without joint expression with SST or GCG) is also greater for phase 7 endocrine cultures than for phase 4 PEC cultures (22.7 versus 12.9 versus 3, 7). Importantly, phase 7 cultures have more cells that only express the INS hormone as a percentage of the total INS population (22.7 / 49.3 = 46% in A and 12.7 / 22.7 = ~ 57 % in B) compared to PEC cultures in phase 4 (3.7 / 15.8 = 23%). Therefore, it seems that phase 7 endocrine cell cultures consist of more cells that express a single hormone, particularly cells that only express INS, than phase 4 PEC cultures. And at least Examples 18 and 20 demonstrate that phase 7 endocrine cells, although still immature, develop and mature to physiologically functional pancreatic islets capable of secreting insulin in response to blood glucose when they are transplanted; while endocrine subpopulations (CHGA +) of phase 4 PEC cultures are not able to do so in vivo. For more information, see FIG. 44 and 45 and the previous examples.

Example 23

ENDOCRINE CELLS (CHGA +) OF PHASE 7 AND PHASE 4 (PEC) DISTINGUISHED BETWEEN YES

Although the analysis of cultures in phase 7 (properly specified endocrine cells) and in phase 4 (PEC) use the same antibodies (for example, CHGA, INS, NKX6.1) for cell staining and analysis, cell subpopulations Endocrine (CHGA +) duly specified in phases 7 and 4 (PEC) are not equal.

Previously, applicants reported that the origin of functional beta cells observed after transplantation of PEC phase 4 cultures was due to pancreatic or non-endocrine progenitor cells (CHGA-) and not primary endocrine cells (CHGA +). Kelly et al. (2011), supra, showed that when PEC cultures were enriched (purified) in endocrine subpopulations (CHGA +) and transplanted, they did not give rise to functional pancreatic islets or beta cells compared to non-enriched PEC cultures. See Kelly et al (2011), page 3-4, FIG. 4 and the complementary Table 1; Schulz et al. (2012) supra; and U.S. patents No. 7,534,608; 7,695,965; 7993920; and 8,278,106. Examples 17 and 18 and Table 19 described a culture of almost pure endocrine cells (CHGA +) (98.2%) in phase 7 which, when transplanted, resulted in a robust functional graft in vivo (FIG. 34). In view of Examples 17 and 18 and other results described herein, the functional population of phase 7 cultures are endocrine cells (CHGA +) in phase 7 and not the significantly lower percentage of non-endocrine cells (CHGA- ) (2.08%). This is in contrast to the PEC phase 4 cultures, about which the applicant had reported in detail in previous disclosures, in which the in vivo function is attributed to the non-endocrine subpopulations (CHGA-). Therefore, in general, endocrine subpopulations in phases 7 and 4 are neither comparable nor equal.

Table 23 shows the flow cytometry data comparing total endocrine subpopulation (CHGA +) and non-endocrine subpopulation (CHGA-) in phase 7 (properly specified endocrine) and phase 4 (PEC), and demonstrates that subpopulations are not equivalent and that distinguish each other. For example, the total percentage of CHGA + subpopulation was significantly higher in phase 7 crops compared to phase 4 crops (85.2 vs. 39.4). Therefore, the total percentage of the CHGA-subpopulation was significantly lower in phase 7 cultures compared to phase 4 cultures (14.8 vs. 60.1). In addition, true endocrine cells, for example, beta cells, not only express CHGA + but also express at least INS and NKX6.1. Therefore, a true endocrine cell expresses CHGA + / INS + / NKX6.1 + (triple positive) and is able to function in vivo. Table 23 shows that phase 7 cultures had almost 5 times more CHGA + / INS + / NKX6.1 + (triple positive) cells than phase 4 cultures (37.8 vs. 7.8); and although CHGA + / INS + / NKX6.1 + subpopulations in phase 4 may appear correctly specified, as discussed above, these endocrine subpopulations in phase 4 do not work in vivo when they are transplanted, while non-endocrine subpopulations (CHGA- ) in phase 4 they ripen and work in vivo. Therefore, applicants refer to phase 7 endocrine cells as "immature endocrine cells" or "immature beta cells" and not as endocrine progenitor / precursor cells, since these cells are evolutionarily compromised to become mature beta cells in alive.

Table 23: Comparison of endocrine (CHGA +) and non-endocrine (CHGA-) cells of phases 7 and 4 (PEC)

Endocrine / non-endocrine cell flow cytometry in Phase 7

Endocrine / non-endocrine cell flow cytometry in Phase 4

Example 24

PURIFICATION OF ENDOCRINE CELLS OF CELL POPULATIONS IN PHASE 7 USING A CINC SENSOR

Beta cell secretory vesicles contain high concentrations of zinc. Zinc accumulates in the vesicles at least by the action of the zinc transporter SLC30A8 (or ZnT8). Zinc-binding fluorescent probes have been used to visualize zinc in cells by absorbing and emitting more light at specific wavelengths when they bind with zinc than without zinc. However, reports show that most probes may not be located correctly to detect zinc within beta cell vesicles. Interestingly, PyDPy1 (or Py1; Chemical Communications, 2011, 47: 7107-9) can enter the vesicles, but has not been used with beta cells till the date. Py1 increases the intensity of fluorescence x50-80 when bound to Zn2 +. For the first time, applicants have demonstrated the production of an endocrine cell or immature beta cell, a population capable of functioning in vivo. Therefore, it is advantageous to have a means to further enrich this population for additional in vitro analysis purposes, to use as a screening tool, or to provide a population of immature enriched beta cells for transplantation.

Endocrine cells or immature beta cells in phase 7 were treated with Py1, and the cells were classified by fluorescence to determine those with high zinc content. A Py1 zinc sensor (customized synthesized by ChemoGenics Biopharma, Research Triangle Park, NC) was resuspended at 10 mM in DMSO and diluted in DPBS (- / -) / 0.25% BSA (buffer A) at concentration 5 micromolar end (staining solution). Aggregates of phase 7 cells differentiated were washed twice with DPBS (- / -) and dissociated with Accumax. Accumax was inactivated with the addition of base medium containing B-27. The resulting cell suspension was filtered through a 40 micron mesh, centrifuged, washed in buffer A and resuspended in staining solution. Staining was continued for 15 minutes, the cells were centrifuged, washed in buffer A and resuspended in buffer A for classification. Cells were classified by flow cytometry in 50% CMRL / FBS at 4 ° C. After classification, the cells were centrifuged and resuspended in RNA isolation buffer.

In the results shown in FIG. 40, the cells encompassed or selected by the polygon are living cells that have increased fluorescence due to the presence of a Py1 sensor attached to zinc. This population of cells was divided into two approximately equal analysis windows and classified into two tubes, called bright and dark according to their fluorescence intensity. RNA was prepared from the cells and subjected to a Nanostring analysis.

As seen in FIG. 42A-C, the dark population is enriched in markers of the beta cell lineage such as INS, IAPP, PDX1, NKX6.1, PAX4, PCSK1, G6PC2, GCK and SLC30A8. This indicates that beta cells or immature beta cells can be purified from a phase 7 cell population using a zinc sensor. A previous experiment determined that 630,000 units of INS mRNA per Nanostring correspond to approximately 49% of INS positive cells by flow cytometry (data not shown). Therefore, a purified dark population that has a Nanostring value for INS of 1,200,000 would correspond to approximately 93% of positive cells in INS (1,200,000 / 630,000 x 49%).

In contrast, the population classified as bright was enriched with markers of the alpha cell lineage such as GCG, ARX and SLC30A8 (FIG. 41, 42, 43). And similar to beta cells, glucagon cells are also known to absorb zinc. Therefore, the Py1 sensor can be used to bind zinc into beta and glucagon cells, but separated or classified by their different fluorescence levels.

Summary of methods of preparation of PEC cultures (phases 1-4) and endocrine cells (phases 1-7)

In summary, the inventions described herein are directed to at least PEC and immature endocrine cells and methods of manufacturing said cells comprising at least phases 1-4 for the production of PEC, and phases 1-7 for endocrine cell production. Figures 43, 44 and 45 are diagrams summarizing certain aspects of the applicant's cellular compositions and DE the production methods described herein.

Applicants have previously reported that endocrine subpopulations (CHGA +) that follow a phase 4 differentiation protocol do not become mature and functional pancreatic islet cells when they are transplanted in vivo. See cell type "EN" after phase 4 in FIG. 43 and 44. These endocrine subpopulations (CHGA +) had an early or premature expression of NGN3 (expression of NGN3 before joint expression of PDX1 and NKX6.1 ). See also Rukstalis et al. (2009), supra. In contrast, non-endocrine subpopulations (CHGA-) of PEC that did not express NGN3 develop and mature in endocrine cells in vivo. This delay in the expression of NGN3 until after in vivo maturation of non-endocrine subpopulations (CHGA-) of PEC was shown in Example 10 (FIG. 15-16) where the combination of Activin, Wnt and Heregulin effectively repressed NGN3 in comparison with the control, and the transplantation of this PEC compared with the control improved the function in vivo. See FIG. 15-16.

Applicants attempted to obtain a properly specified endocrine cell culture that was capable of developing and maturing pancreatic islets in vivo and producing insulin in response to blood glucose levels similar to those observed in all cases with p Ec, specifically subpopulation. endocrine (CHGA-) of PEC. To this end, applicants conducted many iterative experiments to suppress or inhibit the expression of NGN3 in phases 3 and 4. Examples 8 to 11 describe in detail the use of Activin by the applicant alone or in combination with other agents such as Wnt and Heregulin, and at various concentrations, to affect the expression or suppression of NGN3. FIG. 44 also summarizes the effect of Activin in phases 3 and 4.

Once it was demonstrated that the expression of NGN3 could be delayed and that it did not affect the function in vivo, the applicants investigated methods to induce the expression of endocrine markers in phases subsequent to the formation of PEC (phase 4). Examples 11-14 and 16-22 describe the numerous iterations and methods used to optimize culture conditions to produce properly specified endocrine populations that could give rise to an in vivo function . Specifically, the applicant used a phase 5 gamma secretase inhibitor to induce NGN3 expression and endocrine differentiation. The applicant used reagents such as CMRL in phases 6 and 7 to increase endocrine marker expression; BMP to increase INS and PDX1. Figures 44 and 45 summarize and describe these efforts.

It will be appreciated that, initially, the use of multiple methodologies is required to characterize and identify cells (eg, Q-PCR, ICC, flow cytometry analysis, C-peptide assays and the like). After these cells have been fully characterized and identified under certain cell differentiation culture conditions, Q-PCR and Nanostring multiplexed RNA were normally used as unique methods to analyze whether such a cell type had been obtained.

The methods, compositions and devices described herein are currently representative of preferred embodiments, and are illustrative and are not intended to be limitations on the scope of the invention. Experts in the field will come up with changes and other uses that are defined by the scope of the disclosure. Accordingly, it will be apparent to one skilled in the art that variable substitutions and modifications of the invention disclosed herein may be made.

As used in the following claims and throughout this disclosure, the term "consisting essentially of" means that it includes any item listed after the expression and is limited to other items that do not interfere or contribute to the activity or action specified in the disclosure of the items listed. Therefore, the expression "consisting essentially of" indicates that the items listed are necessary or mandatory, but that other items are optional and may or may not be present depending on whether or not they affect the activity or action of the items listed. Likewise, it will be appreciated that, in embodiments in which numerical values are mentioned, such as quantities, concentrations, percentages, proportions or intervals, the referenced value may be "at least approximately" the numerical value, "approximately" the numerical value or "at least" the numerical value.

Claims (10)

1. A method of producing an unipotent human immature beta cell comprising: contacting cells of the definitive human endodermal lineage in vitro with a member of the TGFp superfamily and a member of the Wnt family, thus producing a human immature beta cell Unipotent 2. The method of claim 1, wherein said unipotent human immature beta cell expresses INS, NKX6.1, and does not substantially express NGN3. 3. The method of claim 1, wherein said unipotent immature beta cell expresses INS, NKX6.1 and MAFB, and does not substantially express NGN3. 4. The method of claim 1, further comprising contacting the cells of the definitive human endodermal lineage in vitro with a Heregulin. 5. The method of claim 1, wherein the TGFp superfamily member is Activina A. 6. The method of claim 1, wherein the member of the Wnt family is Wnt3a. 7. The method of claim 4, wherein the Heregulin is Heregulin-1, Heregulin-2, Heregulin-3 or Heregulin-4. 8. The method of claim 1, wherein the cells of the definitive human endodermal lineage are contacted with at least 50 ng / ml of the TGFp superfamily member. 9. The method of claim 1, wherein the cells of the definitive human endodermal lineage are contacted with at least 25 ng / ml of the Wnt family member. 10. The method of claim 4, wherein the cells of the definitive human endodermal lineage are contacted with 5-15 times less Heregulin than the TGFp superfamily member and the Wnt family member.